Patent Publication Number: US-2019185568-A1

Title: Anti-pd-1 antibody

Description:
TECHNICAL FIELD 
     The present invention relates to an anti-PD-1 antibody. More specifically, the present invention relates to an anti-PD-1 antibody comprising a variable region containing complementarily-determining regions (CDRs) of a rat anti-bovine PD-1 antibody and a constant region of an antibody of an animal other than rat. 
     BACKGROUND ART 
     Programmed cell death 1 (PD-1), an immunoinhibitory receptor, and its ligand programmed cell death ligand 1 (PD-L1) are molecules identified by Prof. Tasuku Honjo et al., Kyoto University, as factors which inhibit excessive immune response and are deeply involved in immunotolerance (Non-Patent Document No. 1: Ishida Y, Agata Y, Shibahara K, Honjo T. The EMBO J., 11(11); Nov. 1992). Recently, it has been elucidated that these molecules are also involved in immunosuppression in tumors. In the field of human medical care, an antibody drug that inhibits the effect of PD-1 has been developed and put into practical use (Opdivo™, Ono Pharmaceutical Co., Ltd.). 
     To date, the present inventors have been developing an immunotherapy for animal refractory diseases targeting PD-1 or PD-L 1, and have revealed that this novel immunotherapy is applicable to multiple-diseases and multiple-animals. (Non-Patent Document No. 2: Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K. Vet. Res.; 42: 103; Sep. 2011; Non-Patent Document No. 3: Ikebuchi R, Konnai S; Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Vet. Res., 44: 59; Jul. 22, 2013; Non-Patent Document No. 4: Ikebuchi R, Konnai S, Okagawa T Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology, 142(4): 551-61; Aug. 2014; Non-Patent Document No. 5: Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One, 9(6): e98415; Jun. 10, 2014; Non-Patent Document No. 6: Mingala C N, Konnai S, Ikebuchi R, Ohashi K. Comp. Immunol. Microbiol. Infect. Dis., 34(1): 55-63; Jan. 2011). 
     However, the antibodies which the present inventors have prepared to date are rat antibodies, and therefore it is impossible to administer those antibodies repeatedly to animals other than rat. 
     PRIOR ART LITERATURE 
     Non-Patent Documents 
     Non-Patent Document No. 1: Ishida Y, Agata Y, Shibahara K, Honjo T. The EMBO Journal. 11(11):3887-3895; Nov. 1992 
     Non-Patent Document No. 2: Ikebuchi R, Konnai S, Shirai T, Sunden Y Murata S, Onuma M, Ohashi K. Vet. Res., 42: 103; Sep. 2011.
 
Non-Patent Document No. 3: Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Vet. Res., 44: 59; Jul. 22, 2013.
 
     Non-Patent Document No. 4: Ikehuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y Murata S, Ohashi K. Immunology, 142(4): 551-61; Aug. 2014. 
     Non-Patent Document No. 5: Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One, 9(6): e98415; Jun. 10, 2014.
 
Non-Patent Document No. 6: Mingala C N, Konnai S. Ikebuchi R, Ohashi K. Comp. Immunol. Microbiol. Infect. Dis., 34(1): 55-63; Jan. 2011.
 
     DISCLOSURE OF THE INVENTION 
     Problem for Solution by the Invention 
     It is an object of the present invention to provide an anti-PD-1 antibody capable of repeated administration even to animals other than rat. 
     Means to Solve the Problem 
     The present inventors have determined the variable regions of a rat anti-bovine PD-1 monoclonal antibody (5D2) capable of inhibiting the binding of bovine PD-1 to PD-L1, and then combined genes encoding the resultant variable regions with genes encoding the constant regions of a bovine immunoglobulin (bovine IgG1, with mutations having been introduced into the putative binding sites of Fcγ receptors in CH2 domain in order to inhibit ADCC activity; see  FIGS. 1 and 11  for amino acid numbers and mutations: 251 E→P, 252 L→V, 253 P→A, 254 G→deletion, 348 A→S, 349 P→S; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 Aug; 142(4): 551-561) to thereby obtain a chimeric antibody gene. This gene was introduced into Chinese hamster ovary cells (CHO cells). By culturing/proliferating the resultant cells, the present inventors have succeeded in preparing a rat-bovine chimeric anti-bovine PD-1 antibody Further, the present inventors have determined the CDRs of the variable regions of rat anti-bovine PD-1 monoclonal antibody (5D2). The present invention has been achieved based on these findings. 
     A summary of the present invention is as described below. 
     (1) An anti-PD-1 antibody comprising (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 19) and CDR3 having the amino acid sequence of ARTSSGVEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat.
 
(2) The antibody of (1) above, wherein the light chain variable region and the heavy chain variable region are derived from rat.
 
(3) The antibody of (2) above, wherein the light chain variable region is the light chain variable region of a rat anti-bovine PD-1 antibody and the heavy chain variable region is the heavy chain variable region of a rat anti-bovine PD-1 antibody.
 
(4) The antibody of (3) above, wherein the light chain variable region has the amino acid sequence as shown in SEQ ID NO. 1 and the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO: 2.
 
(5) The antibody of any one of (1) to (4) above, wherein the light chain constant region of an antibody of an animal other than rat has the amino acid sequence of the constant region of lambda chain or kappa chain.
 
(6) The antibody of any one of (1) to (5) above, wherein the heavy chain constant region of an antibody of an animal other than rat has the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4, or has mutations introduced thereinto that reduce ADCC activity and/or CDC activity.
 
(7) The antibody of (6) above, wherein the animal other than rat is bovine; the light chain constant region of the bovine antibody has the amino acid sequence of the constant region of lambda chain; and the heavy chain constant region of the bovine antibody has mutations introduced thereinto that reduce ADCC activity and/or CDC activity.
 
(8) The antibody of (7) above, wherein the light chain constant region of the bovine antibody has the amino acid sequence as shown in SEQ ID NO: 3 and the heavy chain constant region of the bovine antibody has the amino acid sequence as shown in SEQ ID NO: 4
 
(9) The antibody of any one of (1) to (8) above which has a four-chain structure comprising two light chains and two heavy chains.
 
(10) A pharmaceutical composition comprising the antibody of any one of (1) to (9) above as an active ingredient.
 
(11) The composition of (10) above for prevention and/or treatment of cancers and/or inflammations.
 
(12) The composition of (11) above, wherein the cancers and/or inflammations are selected from the group consisting of neoplastic diseases, leukemia, Johne&#39;s disease, anaplasmosis, bacterial mastitis, mycotic mastitis, mycoplasma infections (such as mycoplasma mastitis, mycoplasma pneumonia or the like), tuberculosis,  Theileria orientaiis  infection, cryptosporidiosis, coccidiosis, trypanosomiasis and leishmaniasis.
 
(13) An artificial genetic DNA comprising (a′) a DNA encoding a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NC): 17) and the light chain constant region of an antibody of an animal other than rat and (b′) a DNA encoding a heavy chain comprising a heavy Chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 19) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat.
 
(14) A vector comprising the artificial genetic DNA of (13) above.
 
(15) A host cell transformed with the vector of (14) above.
 
(16) A method of preparing an antibody, comprising culturing the host cell of (15) above and collecting an anti-PD-1 antibody from the resultant culture.
 
(17) A DNA encoding a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat.
 
(18) A DNA encoding a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 19) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat.
 
     Effect of the Invention 
     According to the present invention, a novel anti-PD-1 antibody has been obtained. This antibody is applicable even to those animals other than rat. 
     The present specification encompasses the contents disclosed in the specifications and/or drawings of Japanese Patent Applications No. 2016-159090 and No. 2017-099615 based on which the present patent application claims priority. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       [ FIG. 1 ] The amino acid sequence of rat-bovine chimeric anti-bovine PD-1 antibody ch5D2. CDR1, CDR2 and CDR3 in the light chain and the heavy chain variable regions of rat anti-bovine PD-1 antibody 5D2 are shown. Further, amino acids introduced as mutations to bovine IgG4 (CH2 domain) are also shown (amino acid numbers and mutations: 251 E→P, 252 L→V, 253 P→A, 254 G→deletion, 348 A→S, 349 P→S). 
       [ FIG. 2 ] Schematic drawings of pDN112 vector and rat-bovine chimeric anti-bovine PD-1 antibody ch5D2. 
       [ FIG. 3 ] The amount of production and the purity after purification of rat-bovine chimeric anti-bovine PD-1 antibody ch5D2. 
       [ FIG. 4 ] Binding property of rat-bovine chimeric anti-bovine PD-1 antibody ch5D2. 
       [ FIG. 5 ] Inhibitory activity of rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 against bovine PD-1/PD-L1 binding. 
       [ FIG. 6 ] Transition in blood concentrations of rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 after administration to the cattle experimentally infected with BLV. 
       [ FIG. 7 ] Proliferative response of T cells against BLV antigen in the cattle experimentally infected with BLV through administration of rat-bovine chimeric anti-bovine PD-1 antibody ch5D2. 
       [ FIG. 8 ] Changes in the BLV provirus load in the cattle experimentally infected with BLV through administration of rat-bovine chimeric anti-bovine PD-1 antibody ch5D2. 
       [ FIG. 9 ] Cross-reactivity of rat anti-bovine PD-1 antibody 5D2 with ovine PD-1 
       [ FIG. 10 ] Cross-reactivity of rat anti-bovine PD-1 antibody 5D2 with water buffalo T cells 
       [ FIG. 11 ] 3D structure of bovine IgG1 constant region and putative binding site for Fcγ receptors 
       [ FIG. 12 ] pDC6 vector 
       [ FIG. 13 ] Purities after purification of rat-bovine chimeric anti-bovine PD-1 antibodies ch5D2 IgG1 WT and IgG1 ADCC-. 
       [ FIG. 14 ] Binding of rat-bovine chimeric anti-bovine PD-1 antibodies ch5D2 IgG1 WT and IgG1. ADCC- to individual bovine Fcγ receptors. 
     
    
    
     BEST MODES FOR CARRYING OUT THE INVENTION 
     Hereinbelow, the present invention will be described in detail. 
     The present invention provides an anti-PD-1 antibody comprising light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 19) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat. 
     CDR1, CDR2 and CDR3 in the light chain variable region (VL) of rat anti-bovine PD-1 antibody 5D2 (to be described later) are respectively a region consisting of the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), a region consisting of the amino acid sequence of GVS and a region consisting of the amino acid sequence of FQATHDPDT (SEQ ID NO: 17) (see  FIG. 1 ). 
     Further, CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) of rat anti-bovine PD-1 antibody 5D2 are respectively a region consisting of the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), a region consisting of the amino acid sequence of IRSGGST (SEQ ID NO: 19) and a region consisting of the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) (see  FIG. 1 ). 
     In the amino acid sequences of QSLEYSDGYTY (SEQ ID NO: 16), GVS and FQATHDPDT (SEQ ID NO: 17), as well as the amino acid sequences of GFSLTSYY (SEQ ID NO: 18), IRSGGST (SEQ ID NO: 19) and ARTSSGYEGGFDY (SEQ ID NO: 20), one, two, three, four or five amino acids may be deleted, substituted or added. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function of a CDR in the light chain or heavy chain variable region of the PD-1 antibody. 
     As used herein, the term “antibody” is a concept encompassing not only full-length antibodies but also antibodies of smaller molecular sizes such as Fab, F(ab)′ 2 , ScFv, Diabody, V H , V L , Sc(Fv) 2 , Bispecific sc(Fv) 2 , Minibody, scFv-Fc monomer or scFv-Fc dimer. 
     In the anti-PD-1 antibody of the present invention, the VL and VH may be derived from rat. For example, the VL may be the VL of a rat anti-bovine PD-1 antibody, and the VH may be the VH of the rat anti-bovine PD-1 antibody. 
     The amino acid sequence of the VL and the amino acid sequence of the VH of the rat anti-bovine PD-1 antibody are shown in SEQ ID NOS: 1 and 2, respectively. The amino acid sequences as shown in SEQ ID NOS: 1 and 2 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function as VL or VH of the PD-1 antibody. 
     The VL and VH of an antibody of an animal other than rat may be derived from an animal which produces a PD-1 that cross-reacts with rat anti-bovine PD-1 antibody 5D2. 
     There are two types of immunoglobulin light chain, which are called Kappa chain (κ) and Lambda chain (λ). In the anti-PD-1 antibody of the present invention, the light chain constant region (CL) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of either Kappa chain or Lambda chain. However, the relative abundance of Lambda chain is higher in bovine, ovine, feline, canine and equine, and that of Kappa chain is higher in mouse, rat, human and porcine. Since a chain with a higher relative abundance is considered to be preferable, a bovine, ovine, feline, canine or equine antibody preferably has the amino acid sequence of the constant region of Lambda chain whereas a mouse, rat, human or porcine antibody preferably has the amino acid sequence of the constant region of Kappa chain. 
     The heavy chain constant region (CH) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4. Immunoglobulin heavy chain is classified into γ chain, μ chain, α chain, δ chain and ϵ chain depending on the difference in constant region. According to the type of heavy chain present, five classes (isotypes) of immunoglobulin are format they are IgG, IgM, IgA, IgD and IgE. 
     Immunoglobulin G (IgG) accounts for 70-75% of human immunoglobulins and is the most abundantly found monomeric antibody in plasma. IgG has a four-chain structure consisting of two light chains and two heavy chains. Human IgG1, IgG-2 and IgG4 have a molecular weight of about 146,000, whereas human IgG3 has a long hinge region that connects Fab region and Fc region and has a larger molecular weight of 170,000. Human IgG1 accounts for about 65% of human IgG, human IgG2 about 25%, human IgG3 about 7%, and human IgG4 about 3%. They are uniformly distributed inside and outside of blood vessels. Having a strong affinity for Fc receptors and complement factors on effector cell surfaces, human IgG1 induces antibody-dependent cell cytotoxicity (ADCC) and also activates complements to induce complement-dependent cell cytotoxicity (CDC). Human IgG2 and IgG4 are low at ADCC and CDC activities because their affinity for Fc receptors and complement factors is low. 
     Immunoglobulin M (IgM), which accounts for about 10% of human immunoglobulins, is a pentameric antibody consisting of five basic four-chain structures joined together. It has a molecular weight of 970,000. Usually occurring only in blood, IgM is first produced against infectious microorganisms and takes charge of early stage immunity. 
     Immunoglobulin A (IgA) accounts for 10-15% of human immunoglobulins. It has a molecular weight of 160,000. Secreted IgA is a dimeric antibody consisting of two IgA molecules joined together. IgA1 is found in serum, nasal discharge, saliva and breast milk. In intestinal juice, IgA2 is found abundantly. 
     Immunoglobulin D (IgD) is a monomeric antibody accounting for no more than 1% of human immunoglobulins. IgD is found on B cell surfaces and involved in induction of antibody production. 
     Immunoglobulin E (IgE) is a monomeric antibody that occurs in an extremely small amount, accounting for only 0.001% or less of human immunoglobulins. Immunoglobulin E is considered to be involved in immune response to parasites but in advanced countries where parasites are rare, IgE is largely involved in bronchial asthma and allergy among other things. 
     In canine, sequences of IgG-A (equivalent to human IgG2), IgG-B (equivalent to human IgG1), IgG-C (equivalent to human IgG3) and IgG-D (equivalent to human IgG4) have been identified as the heavy chain of IgG. In the antibody of the present invention, an IgG&#39;s heavy chain constant region with neither ADCC activity nor CDC activity is preferable (IgG4 in human). In the case where the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, one may use a constant region that has lost both ADCC activity and CDC activity as a result of introducing mutations into the relevant region of an immunoglobulin equivalent to human IgG4. 
     In bovine, sequences of IgG1, IgG2 and IgG3 have been identified as the heavy chain of IgG. In the antibody of the present invention, an IgG&#39;s heavy chain constant region with neither ADCC activity nor CDC activity is preferable (IgG4 in human). Although the constant region of wild-type human IgG1 has ADCC activity and CDC activity, it is known that these activities can be reduced by introducing amino acid substitutions or deletions into specific sites. In bovine, the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, so mutations may be added to the relevant region of an immunoglobulin equivalent to human IgG1 and the resultant constant region then used. As one example, the amino acid sequence of the CH of a bovine antibody (IgG1 chain, GenBank: X62916) having mutations introduced into CH2 domain and a nucleotide sequence for such amino acid sequence (after codon optimization) are shown in SEQ ID NOS: 4 and 8, respectively. 
     An anti-PD-1 antibody is more preferable in which (i) the CL of a bovine antibody has the amino acid sequence of the constant region of Lambda chain and (ii) the CH of the bovine antibody has mutations introduced thereinto that reduce ADCC activity and/or CDC activity. 
     The anti-PD-1 antibody of the present invention encompasses rat-bovine chimeric antibodies, bovinized antibodies and complete bovine-type antibodies. However, the animal is not limited to bovine and may be exemplified by human, canine, porcine, simian, mouse, feline, equine, goat, ovine, water buffalo, rabbit, hamster, guinea pig and the like. 
     For example, the anti-PD-1 antibody of the present invention may be an anti-PD-1 antibody in which the CL of a bovine antibody has the amino acid sequence as shown in SEQ ID NO: 3 and the CH of the bovine antibody has the amino acid sequence as shown in SEQ ID NO: 4. 
     The amino acid sequences as shown in SEQ ID NOS: 3 and 4 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function as CL or CH of the PD-1 antibody. 
     The anti-PD-1 antibody of the present invention may have a four-chain structure comprising two light chains and two heavy chains. 
     The anti-PD-1 antibody of the present invention may be prepared as described below. Briefly, an artificial gene is synthesized which comprises (i) the identified variable region sequences of a rat anti-bovine PD-1 antibody and (ii) the constant region sequences of an antibody of an animal other than rat (e.g., bovine) (preferably, an immunoglobulin equivalent to human IgG1, in which mutations have been introduced into the relevant region to reduce ADCC activity and/or CDC activity). The resultant gene is inserted into a vector (e.g., plasmid), which is then introduced into a host cell (e.g., mammal cell such as CHO cell), The host cell is cultured, and the antibody of interest is collected from the resultant culture. 
     The amino acid sequence and the nucleotide sequence of the VL of the rat anti-bovine PD-1 antibody identified by the present inventors are shown in SEQ ID NOS: 1 and 5, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 11. 
     The amino acid sequence and the nucleotide sequence of the VH of the rat anti-bovine PD-1 antibody identified by the present inventors are shown in SEQ ID NOS: 2 and 6, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 12. 
     The amino acid sequence and the nucleotide sequence of the CL (Lambda chain, GenBank: X62917) of a bovine antibody are shown in SEQ ID NOS: 3 and 7, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 13. 
     The amino acid sequence and the nucleotide sequence (after codon optimization) of the CH (IgG1 chain, modified from GenBank: X62916) of a bovine antibody are shown in SEQ ID NOS: 4 and 8, respectively. 
     Further, SEQ ID NO: 9 shows the amino acid sequence of a chimeric light chain consisting of the VL of the rat anti-bovine PD-1 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric light chain consisting of the VL of the rat anti-PD-1 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody is shown in SEQ NO: 14. 
     SEQ ID NO: 10 shows the amino acid sequence of a chimeric heavy chain consisting of the VH of the rat anti-bovine PD-1 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric heavy chain consisting of the VH of the rat anti-bovine PD-1 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody is shown in SEQ ID NO: 15. 
     Amino acid sequences and nucleotide sequences of CLs and CHs of various animals other than rat may be obtained from known databases for use in the present invention. 
     Amino acid sequences and nucleotide sequences of bovine CL and CH are summarized in the table below. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 
               
               
                   
               
               
                   
                   
                   
                   
                 GenBank 
                   
                   
               
               
                   
                   
                   
                   
                 Accession 
                   
                   
               
               
                 Species 
                 Ig Domain  
                 Nucleotide Sequence 
                 Amino Acid Sequence 
                 No.  
                 IMGT Database 
                 Reference 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Bovine 
                 Bovine Ig 
                 IgG1 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC 
                 ASTTAPKVYPLSGGGDKSSSTVTLGC 
                 X62916 
                 http://www. 
                 Symons DB 
               
               
                 (Scientific 
                 heavy chain 
                 variant 1 
                 TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG 
                 LVSSYMPEPVTVTWNSGALKSGVHTFP 
                   
                 imgt.org/ 
                 et al., J 
               
               
                 Name  Bos   
                 constant 
                   
                 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC 
                 AVLQSSGLYSLSSMVTVPGSTSGQTFT 
                   
                 IMGT 
                 Immogenet, 
               
               
                   taurus ) 
                 region 
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA 
                 CNVAHPASSTKVDKAVDPTCKPSPCD 
                   
                 repertoire/ 
                 14, 273- 
               
               
                   
                 (CH1 CH3) 
                   
                 CACCTTCCCGGCTGTCCTTCAGTCCTCCGGGCTGTACT 
                 CCPPPELPGGPSVFIFPPKPKDTLTISG 
                   
                 index.php? 
                 283 (1987) 
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA 
                 TPEVTCVVVDVGHDDPEVKFSWFVDD 
                   
                 section= 
                 PMID: 
               
               
                   
                   
                   
                 GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG 
                 VEVNTATTKPREEQFNSTYRVVSALRI 
                   
                 LocusGenes&amp; 
                 3141517 
               
               
                   
                   
                   
                 CAGCACCAAGGTGGACAAGGCTGTTGATCCCACATGCA 
                 QHQDWTGGKEFKCKVHNEGLPAPTVRT 
                   
                 repertoire= 
                 Symons DB 
               
               
                   
                   
                   
                 AACCATCACCCTGTGACTGTTGCCCACCCCCTGAGTC 
                 ISRTKGPAREPQVYVLAPPQEELSKST 
                   
                 genetable&amp; 
                 et al., 
               
               
                   
                   
                   
                 CCCGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC 
                 VSLTCMVTSFYPDYIAVEWQRNGQPES 
                   
                 species= 
                 Mol. 
               
               
                   
                   
                   
                 CAAGGACACCCTCACAATCTGGGGAACGCCCGAGGTCA 
                 EDKYGTTPPQLDADSSYFLYSKLRVDR 
                   
                 bovine&amp;group= 
                 Immunol., 
               
               
                   
                   
                   
                 CGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAG 
                 NSWQEGDTYTCVVMHEALHNHYTQKS 
                   
                 IGHC 
                 841-850 
               
               
                   
                   
                   
                 GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA 
                 TSKSAGK* 
                   
                   
                 (1989). 
               
               
                   
                   
                   
                 CACAGCCACGAGGAAGCCGAGAGAGGAGCAGTTCAACA 
                 (SEQ ID NO: 21) 
                   
                   
                 PMID: 
               
               
                   
                   
                   
                 GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC 
                   
                   
                   
                 2513487 
               
               
                   
                   
                   
                 CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT 
                   
                   
                   
                 Kacskovica 
               
               
                   
                   
                   
                 CCACAACGAAGGCCTCCCGGCCCCCATCGTGAGGACCA 
                   
                   
                   
                 L. and 
               
               
                   
                   
                   
                 TCTCCAGGACCAAAGGGCCGGCCCGGGAGCCGCAGGT 
                   
                   
                   
                 Butler JE., 
               
               
                   
                   
                   
                 GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA 
                   
                   
                   
                 Mol. 
               
               
                   
                   
                   
                 GCAGGGTCAGCCTCACCTGCATGGTCACCAGCTTCTAC 
                   
                   
                   
                 Immunol., 
               
               
                   
                   
                   
                 CCAGACTACATCGCCGTGGAGTGGCAGAGAAAGGGGCA 
                   
                   
                   
                 33, 189- 
               
               
                   
                   
                   
                 GCCTGAGTCGGAGGACAAGTACGGCACCACCCCGCCC 
                   
                   
                   
                 195 (1996). 
               
               
                   
                   
                   
                 CAGCTGGACGCCGACAGCTCCTACTTCCTGTACAGCAA 
                   
                   
                   
                 PMID: 
               
               
                   
                   
                   
                 GCTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGAC 
                   
                   
                   
                 3649440 
               
               
                   
                   
                   
                 ACCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAA 
                   
                   
                   
                 Rabbani H. 
               
               
                   
                   
                   
                 TCACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTA 
                   
                   
                   
                 et al., 
               
               
                   
                   
                   
                 AATGA 
                   
                   
                   
                 Immuno- 
               
               
                   
                   
                   
                 (SEQ ID NO: 29) 
                   
                   
                   
                 genetics, 
               
               
                   
                   
                 IgG1 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC 
                 ASTTAPKVYPLSSCCGDKSSSTVTLGC 
                 X16701 
                   
                 46. 326- 
               
               
                   
                   
                 variant 2 
                 TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG 
                 LVSSYMPEPVTVTWNSGALKSGVHTFP 
                 (M25278) 
                   
                 331 (1997). 
               
               
                   
                   
                   
                 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC 
                 AVLQSSGLYSLSSMVTVPGSTSGQTFT 
                   
                   
                 PMID: 
               
               
                   
                   
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA 
                 CNVAHPASSTKVDKAVDPTCKPSPCD 
                   
                   
                 9218535 
               
               
                   
                   
                   
                 CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTGTACT 
                 CCPPPELPGGPSVFIFPPKPKDTLTISG 
                   
                   
                 Saini S.S. 
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA 
                 TPEVTCVVVDVGHDDPEVKFSWFVDD 
                   
                   
                 et al., 
               
               
                   
                   
                   
                 GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG 
                 VEVNTATTKPREEQFNSTYRVVSALRI 
                   
                   
                 Scand J. 
               
               
                   
                   
                   
                 CAGCACCAAGGTGGACAAGGCTGTTGATCCCACATGCA 
                 QHQDWTGGKEFKCKVHNEGLPAPIVRT 
                   
                   
                 Immunol. 
               
               
                   
                   
                   
                 AACCATCACCCTGTGACTGTTGCCCACCCCCTGAGCTC 
                 ISRTKGPAREPQVYVLAPPQEELSKST 
                   
                   
                 65, 32-8 
               
               
                   
                   
                   
                 CCCGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC 
                 VSLTCMVTSFYPDYIAVEWQRNGQPES 
                   
                   
                 (2007). 
               
               
                   
                   
                   
                 CAAGGACACCCTCACAATCTCGGGAACGCCCGAGGTCA 
                 EDKYGTTPPQLDADSSYFLYSKLRVDR 
                   
                   
                 PMID: 
               
               
                   
                   
                   
                 CGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAG 
                 NSWQEGDTYTCVVMHEALHNHYTQKS 
                   
                   
                 17212764 
               
               
                   
                   
                   
                 GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA 
                 TSKSAGK* 
                   
                   
                   
               
               
                   
                   
                   
                 CACAGCCACGACGAAGCCGAGAGAGGAGCAGTTCAACA 
                 (SEQ ID NO: 22) 
                   
                   
                   
               
               
                   
                   
                   
                 GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCACAACGAAGGCCTCCCGGCCCCCATCGTGAGGACCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCTCCAGGACCAAAGGGCCGGCCCGGGAGCCGCAGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCACGGTCAGCCTCACCTGCATGGTCACCAGCTTCTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCAGACTACATCGCCGTGGAGTGGCAGAGAAACGGGCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCCTGAGTCGGAGGACAAGTACGGCACGACCCCGCCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAGCTGGACGCCGACAGCTCCTACTTCCTGTACAGCAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 30) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG1 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC 
                 ASTTAPKVYPLSSCCGDKSSSTVTLGC 
                 S82409 
                   
                   
               
               
                   
                   
                 variant 3 
                 TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG 
                 LVSSYMPEPVTVTWNSGALKSGVHTFP 
                   
                   
                   
               
               
                   
                   
                   
                 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC 
                 AVLQSSGLYSLSSMVTVPGSTSGTQTF 
                   
                   
                   
               
               
                   
                   
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA 
                 TCNVAHPASSTKVDKAVDPRCKTTCD 
                   
                   
                   
               
               
                   
                   
                   
                 CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT 
                 CCPPPELPGGPSVFIFPPKPKDTLTISG 
                   
                   
                   
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA 
                 TPEVTCVVVDVGHDDPEVKFSWFVDD 
                   
                   
                   
               
               
                   
                   
                   
                 GGAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC 
                 VEVNTATTKPREEQFNSTYRVVSALRI 
                   
                   
                   
               
               
                   
                   
                   
                 CAGCAGCACCAAGGTGGACAAGGCTGTTGATCCCAGAT 
                 QHQDWTGGKEFKCKVHNEGLPAPIVRT 
                   
                   
                   
               
               
                   
                   
                   
                 GCAAAACAACCTGTGACTGTTGCCCACCGCCTGAGCTC 
                 ISRTKGPAREPQVYVLAPPQEELSKST 
                   
                   
                   
               
               
                   
                   
                   
                 CCTGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC 
                 VSLTCMVTSFYPDYIAVEWQRNGQPES 
                   
                   
                   
               
               
                   
                   
                   
                 CAAGGACACCCTCACAATCTCGGGAACGCCCGAGGTCA 
                 EDKYGTTPPQLDADGSYFLYSRLRVDR 
                   
                   
                   
               
               
                   
                   
                   
                 CGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAG 
                 NSWQEGDTYTCVVMHEALHNHYTQKS 
                   
                   
                   
               
               
                   
                   
                   
                 GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA 
                 TSKSAGK* 
                   
                   
                   
               
               
                   
                   
                   
                 CACAGCCACGACGAAGCCGAGAGAGGAGCAGTTCAACA 
                 (SEQ ID NO: 23) 
                   
                   
                   
               
               
                   
                   
                   
                 GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCACAACGAAGGCCTCCCAGCCCCCATCGTGAGGACCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCTCCAGGACCAAAGGGCCGGCCCGGGAGCCGCAGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCACGGTCAGCCTCACCTGCATGGTCACCAGCTTCTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCAGACTACATCGCCGTGGAGTGGCAGAGAAATGGGCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCCTGAGTCAGAGGACAAGTACGGCACGACCCCTCCCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGCTGGACGCCGACGGCTCCTACTTCCTGTACAGCAGG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAAT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 31) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG2 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC 
                 ASTTAPKVYPLASSCGDTSSSTVTLGC 
                 S82407 
                   
                   
               
               
                   
                   
                 variant 1 
                 CAGCTGCGGAGACACATCCAGCTCCACCGTGACCCTGG 
                 LVSSYMPEPVTVTWNSGALKSGVHTFP 
                   
                   
                   
               
               
                   
                   
                   
                 GCTGCCTGGTGTCCAGCTACATGCCCGAGCCGGTGACC 
                 AVLQSSGLYSLSSMVTVPASSSGQTFT 
                   
                   
                   
               
               
                   
                   
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA 
                 CNVAHPASSTKVDKAVGVSIDCSKCHN 
                   
                   
                   
               
               
                   
                   
                   
                 CACCTTCCCGGCTGTCCTTCAGTCCTCCGGGCTCTACT 
                 QPCVREPSVFIFPPKPKDTLMITGTPEV 
                   
                   
                   
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACCGTGCCCGCCAGCAGCTCA 
                 TCVVVNVGHDNPEVQFSWFVDDVEVH 
                   
                   
                   
               
               
                   
                   
                   
                 GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG 
                 TARSKPREEQFNSTYRVVSALPIQHQD 
                   
                   
                   
               
               
                   
                   
                   
                 CAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCATTG 
                 WTGGKEFKCKVNNKGLSAPIVRISRSK 
                   
                   
                   
               
               
                   
                   
                   
                 ACTGCTCCAAGTGTCATAACCAGCCTTGCGTGAGGGAA 
                 GPAREPQVYVLDPPKEELSKSTLSVTC 
                   
                   
                   
               
               
                   
                   
                   
                 CCATCTGTCTTCATCTTCCCACCGAAACCCAAAGACAC 
                 MVTGFYPEDVAVEWQRNRQTESEDKY 
                   
                   
                   
               
               
                   
                   
                   
                 CCTGATGATCACAGGAACGCCCGAGGTCACGTGTGTGG 
                 RTTPPQLDTDRSYFLYSKLRVDRNSWQ 
                   
                   
                   
               
               
                   
                   
                   
                 TGGTGAACGTGGGCCACGATAACCCCGAGGTGCAGTTC 
                 EGDAYTCVVMHEALHNHYMQKSTSKS 
                   
                   
                   
               
               
                   
                   
                   
                 TCCTGGTTCGTGGATGACGTGGAGGTGCACACGGCCAG 
                 AGK* 
                   
                   
                   
               
               
                   
                   
                   
                 GTCGAAGCCAAGAGAGGAGCAGTTCAACAGCACGTACC 
                 (SEQ ID NO: 24) 
                   
                   
                   
               
               
                   
                   
                   
                 GCGTGGTCAGCGCCCTGCCCATCCAGCACCAGGACTGG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACAACAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGGCCTCTGGGCCCCCATCGTGAGGATCATCTCCAGGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGTCCT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGACCCACCCAAGGAAGAGCTCAGCAAAAGCACGCTCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCGTCACCTGCATGGTCACCGGCTTCTACCCAGAAGAT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTAGCCGTGGAGTGGCAGAGAAACCGGCAGACTGAGTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGAGGACAAGTACCGCACGACCCCGCCCCAGCTGGAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCGACCGCTCCTACTTCCGTACAGCAAGCTCAGGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGACAGGAACAGCTGGCAGGAAGGAGACGCCTACACG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TGTGTGGTGATGCACGAGGCCCTGCACAATCACTACAT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 32) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG2 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC 
                 ASTTAPKVYPLSSCCGDKSSSTVTLGC 
                 M36946 
                   
                   
               
               
                   
                   
                 variant 2 
                 TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG 
                 LVSSYMPEPVTVTWNSGALKSGVHTFP 
                 (X06703) 
                   
                   
               
               
                   
                   
                   
                 GCTGCCTGGTGTCCAGCTACATGCCCGAGCGGGTGACC 
                 AVLQSSGLYSLSSMVTVPGSTSGQTFT 
                   
                   
                   
               
               
                   
                   
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA 
                 CNVAHPASSTKVDKAVGVSSDCSKPN 
                   
                   
                   
               
               
                   
                   
                   
                 CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT 
                 NQHCVREPSVFIFPPKPKDTLMITGTPE 
                   
                   
                   
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA 
                 VTCVVVNVGHDNPEVQFSWFVDDVEV 
                   
                   
                   
               
               
                   
                   
                   
                 GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG 
                 HTARTKPREEQFNSTYRVVSALPIQHQ 
                   
                   
                   
               
               
                   
                   
                   
                 CAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCAGTG 
                 DWTGGKEFKCKVNIKGLSASIVRIISRSK 
                   
                   
                   
               
               
                   
                   
                   
                 ACTGCTCCAAGCCTAATAACCAGCATTGCGTGAGGGAA 
                 GPAREPQVYVLDPPKEELSKSTVSVTC 
                   
                   
                   
               
               
                   
                   
                   
                 CCATCTGTCTTCATCTTCCCACCGAAACCCAAAGACAC 
                 MVIGFYPEDVDVEWQRDRQTESEDKYR 
                   
                   
                   
               
               
                   
                   
                   
                 CCTGATGATCACAGGAACGCCCGAGGTCACGTGTGTGG 
                 TTPPQLDADRSYFLYSKLRVDRNSWQR 
                   
                   
                   
               
               
                   
                   
                   
                 TGGTGAACGTGGGCCACGATAACCCCGAGGTGCAGTTC 
                 GDTYTCVVMHEALHNHYMQKSTSKSA 
                   
                   
                   
               
               
                   
                   
                   
                 TCCTGGTTCGTGGACGACGTGGAGGTGCACACGGCCAG 
                 GK* 
                   
                   
                   
               
               
                   
                   
                   
                 GACGAAGCCGAGAGAGGAGCAGTTCAACAGCACGTACC 
                 (SEQ ID NO: 25) 
                   
                   
                   
               
               
                   
                   
                   
                 GCGTGGTCAGCGCCCTGCCCATCCAGCACCAGGACTGG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACATCAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGGCCTCTCGGCCTCCATCGTGAGGATCATCTCCAGGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGTCCT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGACCCACCCAAGGAAGAGCTCAGCAAAAGCACGGTCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCGTCACCTGCATGGTCATCGGCTTCTACCCAGAAGAT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTAGACGTGGAGTGGCAGAGAGACCGGCAGACTGAGTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGAGGACAAGTACCGCACGACCCCGCCCCAGCTGGAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCCGACCGCTCCTACTTCCTGTACAGCAAGCTCAGGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGACAGGAACAGCTGGCAGAGAGGAGACACCTACACGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTGTGGTGATGCACGAGGCCCTGCACAATCACTACATG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 33) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG2 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC 
                 ASTTAPVKYPLSSCCGDKSSSGVTLGC 
                 X16702 
                   
                   
               
               
                   
                   
                 variant 3 
                 TTGCTGCGGGGACAAGTCCAGCTCGGGGGTGACCCTGG 
                 LVSSYPEPVTVTWNSGALKSGVHTFP 
                 (M25279) 
                   
                   
               
               
                   
                   
                   
                 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC 
                 AVLQSSGLYSLSSMVTVPASSSGTQTF 
                   
                   
                   
               
               
                   
                   
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA 
                 TCNVHAPASSTKVDKAVGVSSDCSKP 
                   
                   
                   
               
               
                   
                   
                   
                 CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT 
                 NNQHCVREPSVFIFPPKPKDTLMITGTP 
                   
                   
                   
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACCGTGCCCGCCAGCAGCTCA 
                 EVTCVVVNVGHDNPEVQFSWFVDDVE 
                   
                   
                   
               
               
                   
                   
                   
                 GGAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC 
                 VHTARTKPREEQFNSTYRVVSALPIQH 
                   
                   
                   
               
               
                   
                   
                   
                 CAGCAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCA 
                 QDWTGGKEFKCKVNIKGLSASIVRIISRS 
                   
                   
                   
               
               
                   
                   
                   
                 GTGACTGCTCCAAGCCTAATAACCAGCATTGCGTGAGG 
                 KGPAREPQVYVLDPPKEELSKSTVSLT 
                   
                   
                   
               
               
                   
                   
                   
                 GAACCATCTGTCTTCATCTTCCCACCGAAACCCAAAGA 
                 CMVIGFYPEDVDVEWQRDRQTESEDKY 
                   
                   
                   
               
               
                   
                   
                   
                 CACCCTGATGATCACAGGAACGCCCGAGGTCACGTGTG 
                 RTTPPQLDADRSYFLYSKLRVDRNSWQ 
                   
                   
                   
               
               
                   
                   
                   
                 TGGTGGTGAACGTGGGCCACGATAACCCCGAGGTGCAG 
                 RGDTYTCVVMHEALHNHYMQKSTSKS 
                   
                   
                   
               
               
                   
                   
                   
                 TTCTCCTGGTTCGTGGACGACGTGGAGGTGCACACGGC 
                 AGK* 
                   
                   
                   
               
               
                   
                   
                   
                 CAGGACGAAGCCGAGAGAGGAGCAGTTCAACAGCACGT 
                 (SEQ ID NO: 26) 
                   
                   
                   
               
               
                   
                   
                   
                 ACCGCGTGGTCAGCGCCCTGCCCATCCAGCACCAGGAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TGGACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACAT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAAAGGCCTCTCGGCCTCCATCGTGAGGATCATCTCCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGAGCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCTGGACCCACCCAGGAAGAGCTCAGCAAAAGCACGG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCAGCCTCACCTGCATGGTCATCGGCTTCTACCCAGAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GATGTAGACGTGGAGTGGCAGAGAGACCGGCAGACTGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTCGGAGGACAAGTACCGCACGACCCCGCCCCAGCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GACGCCGACCGCTCCTACTTCCTGTACAGCAAGCTCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGTGGACAGGAACAGCTGGCAGAGAGGAGACACCTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACGTGTGTGGTGATGCACGAGGCCCTGCACAATCACTA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CATGCAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 34) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG3 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC 
                 ASTTAPKVYPLASSCGDTSSSTVTLGC 
                 U63638 
                   
                   
               
               
                   
                   
                 variant 1 
                 CAGCTGCGGAGACACATCCAGCTCCACCGTGACCCTGG 
                 LVSSYMPEPVTVTWNSGALKSGVHTFP 
                   
                   
                   
               
               
                   
                   
                   
                 GCTGCCTGGTCTCCAGCTACATGCCCGAGGGGGTGACC 
                 AVRQSSGLYSLSSMVTVPASSSETQTF 
                   
                   
                   
               
               
                   
                   
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA 
                 TCNVAHPASSTKVDKAVTARRPVPTTP 
                   
                   
                   
               
               
                   
                   
                   
                 CACCTTCCCGGCCGTCCGGCAGTCCTCTGGGCTGTACT 
                 KTTIPPGKPTTPKSEVEKTPCQCSKCP 
                   
                   
                   
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACTGTGCCCGCCAGCAGCTCA 
                 EPLGGLSVFIFPPKPKDTLTISGTPEVT 
                   
                   
                   
               
               
                   
                   
                   
                 GAAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC 
                 CVVVDVGQDDPEVQFSWFVDDVEVHT 
                   
                   
                   
               
               
                   
                   
                   
                 CAGCAGCACCAAGGTGGACAAGGCTGTCACTGCAAGGC 
                 ARTKPREEQFNSTYRVVSALRIQHQDW 
                   
                   
                   
               
               
                   
                   
                   
                 GTCCAGTCCCGACGACGCCAAAGACAACTATCCCTCCT 
                 LQGKEFKCKVNNKGLPAPIVRTISRTKG 
                   
                   
                   
               
               
                   
                   
                   
                 GGAAAACCCACAACCCCAAAGTCTGAAGTTGAAAAGAC 
                 QAREPQVYVLAPPREELSKSTLSLTCLI 
                   
                   
                   
               
               
                   
                   
                   
                 ACCCTGCCAGTGTTCCAAATGCCCAGAACCTCTGGGAG 
                 TGFYPEEIDVEWQRNGQPESEDKYHITT 
                   
                   
                   
               
               
                   
                   
                   
                 GACTGTCTGTCTTCATCTTCCCACCGAAACCCAAGGAC 
                 APQLDADGSYFLYSKLRVNKSSWQEG 
                   
                   
                   
               
               
                   
                   
                   
                 ACCCTCACAATCTCGGGAACGCCCGAGGTCAGGTGTGT 
                 DHYTCAVMHEALRNHYKEKSISRSPGK 
                   
                   
                   
               
               
                   
                   
                   
                 GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAG 
                 * 
                   
                   
                   
               
               
                   
                   
                   
                 TTCTCCTGGTTCGTGGACGACGTGGAGGTGCACACGGC 
                 (SEQ ID NO: 27) 
                   
                   
                   
               
               
                   
                   
                   
                 CAGGACGAAGCCGAGAGAGGAGCAGTTCAACAGCACCT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCGCGTGGTCAGCGCCCTGCGCATCCAGCACCAGGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTGGCTGCAGGGAAAGGAGTTCAAGTGCAAGGTCAACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACAAAGGCCTCCCGGCCCCCATTGTGAGGACCATCTCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTATG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCCTGGCCCCACCCCGGGAAGAGCTCAGCAAAGCACG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTCAGCCTCACCTGCCTGATCACCGGTTTCTACCCAGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGAGATAGACGTGGAGTGGCAGAGAAATGGGCAGCCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGTCGGAGGACAAGTACCACACGACCGCACCCCAGCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GATGCTGACGGCTCCTACTTCCTGTACAGCAAGCTCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGTGAACAAGAGCAGCTGGCAGGAAGGAGACCACTACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CGTGTGCAGTGATGCACGAAGCTTTACGGAATCACTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAAGAGAAGTCCATCTCGAGGTCTCCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 35) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG3 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC 
                 ASTTAPKVYPLASRCGDTSSSTVTLGC 
                 U63639 
                   
                   
               
               
                   
                   
                 variant 2 
                 CCGCTGCGGAGACACATCCAGCTCCACCGTGACCCTGG 
                 LVSSYMPEPVTVTWNSGALKSGVHTFP 
                   
                   
                   
               
               
                   
                   
                   
                 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC 
                 AVLQSSGLYSLSSMVTVPASTSETQTF 
                   
                   
                   
               
               
                   
                   
                   
                 GTGACCTGGAACTCGGGTGCCCTGAAGAGTGGCGTGCA 
                 TCNVAHPASSTKVDKAVTARRPVPTTP 
                   
                   
                   
               
               
                   
                   
                   
                 CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTGTACT 
                 KTTIPPGKPTTQESEVEKTPCQCSKCP 
                   
                   
                   
               
               
                   
                   
                   
                 CTCTCAGCAGCATGGTGACCGTGCCCGCCAGCACCTCA 
                 EPLGGLSVFIFPPKPKDTLTISGTPEVT 
                   
                   
                   
               
               
                   
                   
                   
                 GAAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC 
                 CVVVDVGQDDPEVQFSWFVDDVEVHT 
                   
                   
                   
               
               
                   
                   
                   
                 CAGCAGCACCAAGGTGGACAAGGCTGTCACTGCAAGGC 
                 ARTKPREEQFNSTYRVVSALRIQHQDW 
                   
                   
                   
               
               
                   
                   
                   
                 GTCCAGTCCCGACGACGCCAAAGACAACCATCCCTCCT 
                 LQGKEFKCKVNNKGLPAPIVRTISRTKG 
                   
                   
                   
               
               
                   
                   
                   
                 GGAAAACCCACAACCCAGGAGTCTGAAGTTGAAAAGAC 
                 QAREPQVYVLAPPPREELSKSTLSLTCLI 
                   
                   
                   
               
               
                   
                   
                   
                 ACCCTGCCAGTGTTCCAAATGCCCAGAACCTCTGGGAG 
                 TGFYPEEIDVEWQRNGQPESEDKYHTT 
                   
                   
                   
               
               
                   
                   
                   
                 GACTGTCTGTCTTGATCTTCCCACCGAAACCCAAGGAC 
                 APQLDADGSYFLYSRLRVNKSSWQEG 
                   
                   
                   
               
               
                   
                   
                   
                 ACCCTCACAATCTCGGGAACGCCCGAGGTCACGTGTGT 
                 DYYTCAVMHEALRNHYKEKSISRSPGK 
                   
                   
                   
               
               
                   
                   
                   
                 GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAG 
                 * 
                   
                   
                   
               
               
                   
                   
                   
                 TTCTCCTGGTTGGTGGACGACGTGGAGGTGCACACGGC 
                 (SEQ ID NO: 28) 
                   
                   
                   
               
               
                   
                   
                   
                 CAGGACGAAGCCGAGAGAGGAGCAGTTCAACAGCACCT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCGCGTGGTCAGCGCCCTGCGCATCCAGCACCAGGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTGGCTGCAGGGAAAGGAGTTCAAGTGCAAGGTCAACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACAAAGGCCTCCCGGCCCCCATTGTGAGGACCATCTCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTATG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCCTGGCCCCACCCCGGGAAGAGCTCAGCAAAAGCACG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTCAGCCTCACCTGCCTGATCACCGGTTTCTACCCAGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGAGATAGACGTGGAGTGGCAGAGAAATGGGCAGCCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGTCGGAGGACAAGTACCACACGACCGCACCCCAGCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GATGCTGACGGCTCCTACTTCCTGTACAGCAGGCTCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGTGAACAAGAGCAGCTGGCAGGAAGGAGACCACTACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CGTGTGCAGTGATGCATGAAGCTTTACGGAATCACTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAAGAGAAGTCCATCTCGAGGTCTCCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 36) 
                   
                   
                   
                   
               
               
                   
                 Bovine Ig 
                 Ig lambda 
                 CAGCCCAAGTCCCCACCCTCGGTCACCCTGTTCCCGCC 
                 QPKSPPSVTLFPPSTEELNGNKATLVC 
                 X62917 
                 Not 
                 Chen L. 
               
               
                   
                 light chain 
                   
                 CTCCACGGAGGAGCTCAACGGCAACAAGGCCACCCTG 
                 LISDFYPGSVTVVWKADGSTITRNVETT 
                   
                 registered 
                 et al., 
               
               
                   
                 constant 
                   
                 GTGTGTCTCATCAGCGACTTCTACCCGGGTAGCGTGAC 
                 RASKQSNSKYAASSYLSLTSSDWKSKG 
                   
                   
                 Vet, 
               
               
                   
                 region (CL) 
                   
                 CGTGGTCTGGAAGGCAGACGGCAGCACCATCACCCGCA 
                 SYSCEVTHEGSTVTKTVKPSECS* 
                   
                   
                 Immunol. 
               
               
                   
                   
                   
                 ACGTGGAGACCACCCGGGCCTCCAAACAGAGCAACAG 
                   
                   
                   
                 Immuno- 
               
               
                   
                   
                   
                 CAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGAGCA 
                   
                   
                   
                 pathol. 
               
               
                   
                   
                   
                 GCGACTGGAAATCGAAAGGCAGTTACAGCTGCGAGGTC 
                   
                   
                   
                 124, 284- 
               
               
                   
                   
                   
                 ACGCACGAGGGGAGCACCGTGACGAAGACAGTGAAGC 
                   
                   
                   
                 294 (2008). 
               
               
                   
                   
                   
                 CCTCAGAGTGTTCTTAG 
                   
                   
                   
                 PMID: 
               
               
                   
                   
                   
                 (SEQ ID NO: 7) 
                   
                   
                   
                 1853886 
               
               
                   
               
            
           
         
       
     
     Amino acid sequences and nucleotide sequences of ovine, water buffalo and human CL and CH are summarized in the table below. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 
               
               
                   
               
               
                   
                   
                   
                   
                 GenBank 
                   
                   
               
               
                   
                   
                   
                   
                 Accession 
                   
                   
               
               
                 Species 
                 Ig Domain  
                 Nucleotide Sequence 
                 Amino Acid Sequence 
                 No.  
                 IMGT Database 
                 Reference 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Ovine 
                 Ovine Ig 
                 IgG1 
                 GCCTCAACAACACCGGCGAAAGTCTACCCTCTGACT 
                 ASTTPPKVYPLTSCCGDTSSSIVTLGC 
                 X69797 
                 http://www. 
                 Dufour V. 
               
               
                 (Scientific 
                 heavy chain 
                   
                 TCTTGCTGCGGGGACACGTCCAGCTCCATCGTGACC 
                 LVSSYMPEPVTVWTNSGALTSGVHTF 
                   
                 IMGT 
                 et al., 
               
               
                 Name  Ovis   
                 constant 
                   
                 CTGGGCTGCCTGGTCTCCAGCTATATGCCCGAGGGG 
                 PAILQSSGLYSLSSVVTVPASTSGAQT 
                   
                 repertoire 
                 J.  
               
               
                   aries ) 
                 region 
                   
                 GTGACCGTGACCTGGAACTCTGGTGCCCTGACCAGC 
                 FICNVAHPASSTKVDKRVEPGCPDPCK 
                   
                 indes.php? 
                 Immunol., 
               
               
                   
                 (CH1 CH3) 
                   
                 GGCGTGCACACCTTCCCGGCCATCCTGCAGTCCTCC 
                 HCRCPPPELPGGPSVFIFPPKPKDTLTI 
                   
                 section= 
                 156, 2163- 
               
               
                   
                   
                   
                 GGGCTCTACTCTCTCAGCAGCGTGGTGACCGTGCCG 
                 SGTPEVTCVVVDVGQDDPEVQFSWFV 
                   
                 LocusGenes&amp; 
                 2170 
               
               
                   
                   
                   
                 GGGCTCTACTCTCTCAGCAGCGTGGTGACCGTGCCG 
                 DNVEVRTARTKPREEQFNSTFRVVSAL 
                   
                 repertoire= 
                 (1996). 
               
               
                   
                   
                   
                 GCCAGCACCTCAGGAGCCCAGACCTTCATGTGCAAC 
                 PIQHQDWTGGKEFKCKVHNEALPAPIV 
                   
                 genetable&amp; 
                 PMID: 
               
               
                   
                   
                   
                 GTAGGCGACCCGGCCAGCAGCACCAAGTGGACAAG 
                 RTISRTKGQAREPQVYVLAPPQEESK 
                   
                 species= 
                 8690905 
               
               
                   
                   
                   
                 CGTGTTGAGCCCGGATGCCCGGACCCATGCAAACAT 
                 STLSVTCLVTFGYPDYIAVEWQKNGQP 
                   
                 sheep&amp;group= 
                   
               
               
                   
                   
                   
                 TGCCGATGCCCACCCCCTGAGCTCCCCGGAGGACC 
                 ESEDKYGTTTSQLDADGSYFLYSRLRV 
                   
                 IGHC 
                   
               
               
                   
                   
                   
                 GTCTGTCTTCATCTTCCCACCGAAACCCAAGGACAC 
                 DKNSWQEGDTYACVVMHEALHNHYTQ 
                   
                   
                   
               
               
                   
                   
                   
                 CCTTACAATCTCTGGAACGCCCGAGGTCACGTGTGT 
                 KSISKPPGK* 
                   
                   
                   
               
               
                   
                   
                   
                 GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGC 
                 (SEQ ID NO: 37) 
                   
                   
                   
               
               
                   
                   
                   
                 AGTTCTCCTGGTTCGTGGACAACGTGGAGGTGCGCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CGGCCAGGACAAAGCCGAGAGAGGAGCAGTTCAACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCACCTTCCGCGTGGTCAGCGCCCTGCCCATCCAGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCAAGACTGGACTGGAGGAAAGGAGTTCAAGTGCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGGTCCACAACGAAGCCCTCCCGGCCCCCATCGTGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGACCATCTCCAGGACCAAAGGGCAGGCCCGGGAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCGCAGGTGTACGTCCTGGCCCCACCCCAGGAAGAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTCAGCAAAAGCACGCTCAGCGTCACCTGCCTGGTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCGGCTTCTACCCAGACTACATCGCCGTGGAGTGG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAGAAAAATGGGCAGCCTGAGTCGGAGGACAAGTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGCACGACCACATCCCAGCTGGACGCCGACGGCTCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TACTTCCTGTACAGCAGGCTCAGGGTGGACAAGAAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGCTGGCAAGAAGGAGACACCTACGCGTGTGTGGTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ATGCACGAGGCTCTGCACAACCACTACACACAGAAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCGATCTCTAAGCCTCCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 38) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG2 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGACT 
                 ASTTAPKVYPLTSCCGDTSSSSSIVTL 
                 X70983 
                   
                 Clarkson 
               
               
                   
                   
                   
                 TCTTGCTGCGGGGACACGTCCAGCTCCAGOTCCATC 
                 CLVSSYMPEPVTVTWNSGALTSGVH 
                   
                   
                 C.A. et 
               
               
                   
                   
                   
                 GTGACCCTGGGCTGCCTGGTCTCCAGCTATATGCCC 
                 TFPAILQSSGLYSLSSVVTVPASTSGA 
                   
                   
                 al., Mol. 
               
               
                   
                   
                   
                 GAGCCGGTGACCGTGACCTGGAACTCTGGTGCCCTG 
                 QTFICNVAHPASSAKVDKRVGISSDYS 
                   
                   
                 Immunol, 
               
               
                   
                   
                   
                 ACCAGCGGCGTGCACACCTTCCCGGCCATCCTGCAG 
                 KCSKPPCVSRPSVFIFPPKPKDSLMITG 
                   
                   
                 30, 1195- 
               
               
                   
                   
                   
                 TCCTCCGGGCTCTACTCTCTCAGCAGCGTGGTGACC 
                 TPEVTCVVVDVGQGDPEVQFSWFVDN 
                   
                   
                 1204 
               
               
                   
                   
                   
                 GTGCCGGCCAGCACCTCAGGAGCCCAGACCTTCATC 
                 VEVRTARTKPREEQFNSTFRVVSALPI 
                   
                   
                 (1993). 
               
               
                   
                   
                   
                 TGCAACGTAGCCCACCCGGCCAGCAGCGCCAAGGTG 
                 QHDHWTGGKEFKCKVHSKGLPAPIVRT 
                   
                   
                 PMID: 
               
               
                   
                   
                   
                 GACAAGCGTGTTGGGATCTCCAGTGACTACTCCAAG 
                 ISRAKGQAREPQVYVLAPPQEELSKST 
                   
                   
                 8413324 
               
               
                   
                   
                   
                 TGTTCTAAACCGCCTTGCGTGAGCCGACCGTCTGTC 
                 LSVTCLVTGFYPDYIAVEWQRARQPES 
                   
                   
                   
               
               
                   
                   
                   
                 TTCATCTTCCCCCCGAAACCCAAGGACAGCCTCATG 
                 EDKYGTTTSQLDADGSYFLYSRLRVDK 
                   
                   
                   
               
               
                   
                   
                   
                 ATCACAGGAACGCCCGAGCTCACGTGTGTGGTGGTG 
                 SSWQRGDTYACVVMHEALHNHYTQKS 
                   
                   
                   
               
               
                   
                   
                   
                 GACGTGGGCCAGGGTGACCCCGAGGTGCAGTTCTCC 
                 ISKPPGK* 
                   
                   
                   
               
               
                   
                   
                   
                 TGGTTCGTGGACAACGTGGAGGTGCGCACGGCCAGG 
                 (SEQ ID NO: 39) 
                   
                   
                   
               
               
                   
                   
                   
                 ACAAAGCCGAGAGAGGAGCAGTTCAACAGCACCTTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CGCGTGGTCAGCGCCCTGCCCATCCAGCACGACCAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TGGACTGGAGGAAAGGAGTTCAAGTGCAAGGTCCAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TAGCAAAGGCCTCCCGGCCCCCATCGTGAGGACCATC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCCAGGGCCAAAGGGCAGGCCCGGGAGCCGCAGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTACGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAGCACGCTCAGCGTCACCTGCCTGGTCACCGGCTT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTACCCAGACTACATCGCCGTGGAGTGGCAGAGAGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCGGCAGCCTGAGTCGGAGGACAAGTACGGCACGAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CACATCCCAGCTGGACGCCGACGGCTCCTACTTCCT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTACAGCAGGCTCAGGGTGGACAAGAGCAGCTGGCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAGAGGAGACACCTACGCCTGTGTCGTGATGCACGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGCTCTGCACAACCACTACACACAGAAGTCGATCTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TAAGCCTCCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 40) 
                   
                   
                   
                   
               
               
                   
                 Ovine Ig 
                 Ig kappa 
                 CCATCCGTCTTCCTCTTCAAACCATCTGAGGAACAG 
                 PSVFLFKPSEEQLRTGTVSVVCLVNDF 
                 X54110 
                 Not 
                 Jenne C.N. 
               
               
                   
                 light chain 
                 (CK) 
                 CTGAGGACCCGAACTGTCTCTGTCGTGTGCTTGGTG 
                 YPKDINVKVKVDGVTQNSNFQNSFTDQ 
                   
                 registered 
                 et al., 
               
               
                   
                 constant 
                   
                 AATGATTTCTACCCCAAAGATATCAATGTCAAGGTGA 
                 DSKKSTYSLSSTLTLSSSEYQSHNAYA 
                   
                   
                 Dev. 
               
               
                   
                 region 
                   
                 AAGTGGATGGGGTTACCCAGAACAGCAACTTCCACA 
                 CEVSHKSLPTALVKSFNKNEC* 
                   
                   
                 Comp. 
               
               
                   
                   
                   
                 ACAGCTTCACAGACCAGGACAGCAAGAAAAGCACCT 
                 (SEQ ID NO: 41) 
                   
                   
                 Immunol. 
               
               
                   
                   
                   
                 ACAGCCTCAGCAGCACCCTCACACTGTCCAGCTCAG 
                   
                   
                   
                 30 (1-2). 
               
               
                   
                   
                   
                 AGTACCAGAGCCATAACGCCTATGCGTGTGAGGTCA 
                   
                   
                   
                 165-174 
               
               
                   
                   
                   
                 GCCACAAGAGCCTGCCCACCGCCCTCGTCAAGAGCT 
                   
                   
                   
                 (2006). 
               
               
                   
                   
                   
                 TCAATAAGAATGAATGTTAG 
                   
                   
                   
                 PMID: 
               
               
                   
                   
                   
                 (SEQ ID NO: 42) 
                   
                   
                   
                 16083958 
               
               
                   
                   
                 Ig lambda 
                 GGTCAGCCCAAGTCCGCACCCTCGGTCACCCTGTTC 
                 GQPKSAPSVTLFPPSTEELSTNKATVV 
                 AY734681 
                   
                   
               
               
                   
                   
                 (CL) 
                 CCGCCTTCCACGGAGCAGCTCAGTACCAACAAGGCC 
                 CLINDFYPGSVNVVWKADGSTINQNVK 
                   
                   
                   
               
               
                   
                   
                   
                 ACCGTGGTCTGTCTCATCAACGACTTCTACCCGGGT 
                 TTQASKQSNSKYAASSYLTLTGSEWKS 
                   
                   
                   
               
               
                   
                   
                   
                 AGCGTGAACGTGGTCTGGAAGGCAGATGGCAGCACC 
                 KSSYTCEVTHEGSTVTKTVKPSECS* 
                   
                   
                   
               
               
                   
                   
                   
                 ATCAATCAGAACGTGAAGACCACCCAGGCCTCCAAA 
                 (SEQ ID NO: 43) 
                   
                   
                   
               
               
                   
                   
                   
                 CAGAGCAACAGCAAGTACGCGGCCAGCAGCTACCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCCTGACGGGCAGCGAGTGGAAGTCTAAGAGCAGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TACACCTGCGAGGTCACGCACGAGGGGAGCACCGTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACGAAGACAGTGAAGCCCTCAGAGTGTTCTTAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 44) 
                   
                   
                   
                   
               
               
                   
               
               
                 Water 
                 Water 
                 IgG1? 
                 GAGCGGCGTGCACACCTTCCCGGCCGTCCTTCAGTCC 
                 SGVHTFPAVLQSSGLYSLSSTVTAPAS 
                 NW_ 
                 Not 
                 None 
               
               
                 buffalo 
                 buffalo Ig 
                   
                 TCCGGGCTCTACTCTCTCAGCAGCACGGTGACCGCGC 
                 ATKSQTFTCNVAHPASSTKVDKAVVP 
                 005690903 
                 registered 
                   
               
               
                 (Scientific 
                 heavy chain 
                   
                 CCGCCAGCGCCACAAAAAGCCAGACCTTCACCTGCAA 
                 PCRPKPCDCCPPPELPGGPSVFIFPPK 
                   
                   
                   
               
               
                 Name: 
                 constant 
                   
                 GGTAGCCCACCCGGCCAGCAGCACCAAGGTGGACAAG 
                 PKDTLTISGTPEVTCVVVDVGHDDPEV 
                   
                   
                   
               
               
                 
                   Bubalus 
                 
                 region 
                   
                 GCTGTTGTTCCCCCATGCAGACCGAAACCCTGTGATTG 
                 KFSWFVDDVEVNTARTKPREEQFNSTY 
                   
                   
                   
               
               
                   bablis ) 
                 (CH1 CH3) 
                   
                 CTGCCCACCCCCTGAGCTCCCCGGAGGACCCTCTGTC 
                 RVVSALPIQHNDWTGGKEFKCKVYNEG 
                   
                   
                   
               
               
                   
                   
                   
                 TTCATCTTCCCACCAAAACCCAAGGACACCCTCACAAT 
                 LPAPIVRTISRTKGQAREPQVYVLAPP 
                   
                   
                   
               
               
                   
                   
                   
                 CTCTGGAACTCCTGAGGTCACGTGTGTGGTGGTGGAC 
                 QDELSKSTVSITCMVTGFYPDYIAVEW 
                   
                   
                   
               
               
                   
                   
                   
                 GTGGGCCACGATGACCCCGAGGTGAAGTTCTCCTGGT 
                 QKDGQPESEDKYGTTPPQLDSDGSYF 
                   
                   
                   
               
               
                   
                   
                   
                 TCGTGGACGATGTGGAGGTAAACACAGCCAGGACGAA 
                 LYSRLRVNKNSWQEGGAYTCVVMHE 
                   
                   
                   
               
               
                   
                   
                   
                 GCCAAGAGAGGAGCAGTTCAACAGCACCTACCGCGTG 
                 (SEQ ID NO: 45) 
                   
                   
                   
               
               
                   
                   
                   
                 GTCAGCGCCCTGCCCATCCAGCACAACGACTGGACTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GAGGAAAGGAGTTCAAGTGCAAGGTCTACAATGAAGGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTCCCAGCCCCCATCGTGAGCACCATCTCCAGCACCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAGGGCAGGCCCGGGAGCCGCAGGTGTACGTCCTGGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCCACCCCAGGACGAGCTCAGCAAAAGCACGGTCAGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ATCACTTGCATGGTCACTGGCTTCTACCCAGACTACAT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CGCCGTAGAGTCGCAGAAAGATGGGCAGCCTGAGTCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GAGGACAAATATGGCACGACCCCGCCCCAGCTGGACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCGATGGCTCCTACTTCCTGTACAGCAGGCTCAGGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GAACAAGAACAGCTGGCAAGAAGGAGGCGCCTACACG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TGTGTAGTGATGCATGAGGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 46) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG2? 
                 GCCTCCATCACAGCCCCGAAAGTCTACCCTCTGACTTC 
                 ASITAPKVYPLTSCRGETSSSTVTLGC 
                 NW_ 
                   
                   
               
               
                   
                   
                   
                 TTGCCGCGGGGAAACGTCCACCTCCACCGTGACCCTG 
                 LVSSYMPEPVTVTWNSGALKSGVHTF 
                 005766143 
                   
                   
               
               
                   
                   
                   
                 GGCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGA 
                 TAVLQSSGLYSLSSTVTAPASATKSQT 
                   
                   
                   
               
               
                   
                   
                   
                 CCGTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGT 
                 FTCNVAHPASSTKVDTAVGFSSDCCK 
                   
                   
                   
               
               
                   
                   
                   
                 GCACACCTTCCCCGCCGTCCTTCAGTCCTCTGGGCTC 
                 FPKPCVRGPSVFIFPPKPKDTLMITGNP 
                   
                   
                   
               
               
                   
                   
                   
                 TACTCTCTCAGCAGCACGGTGACCGCGCCCGCCAGCG 
                 EVTCVVVDVGRDNPEVQFSWFVGDVE 
                   
                   
                   
               
               
                   
                   
                   
                 CCACAAAAAGCCAGACCTTCACCTGCAACGTAGCCCAC 
                 VHTGRSKPREEQFNSTYRVVSTLPIQH 
                   
                   
                   
               
               
                   
                   
                   
                 CCGGCCAGCAGCACCAAGGTGGACACGGCTGTTGGGT 
                 NDWTGGKEFKCKVNNKGLPAPIVRTIS 
                   
                   
                   
               
               
                   
                   
                   
                 TCTCCAGTGACTGCTCCAAGTTTCCTAAGCCTTGTGTG 
                 FTKGQAREPQVYVLAPPQEELSKSTVS 
                   
                   
                   
               
               
                   
                   
                   
                 AGGGGACCATCTGTCTTCATCTTCCCGCCGAAACCCAA 
                 VTCMVTGFYPDYIAVEWHRDRQAESED 
                   
                   
                   
               
               
                   
                   
                   
                 AGACACCCTGATGATCACAGGAAATCCCGAGGTCACAT 
                 KYRTTPPQLDSDGSYFLYSRLKVNKNS 
                   
                   
                   
               
               
                   
                   
                   
                 GTGTGGTGGTGGACGTGGGCCGGGATAACCCCGAGGT 
                 GQEGGAYTCVVMHE 
                   
                   
                   
               
               
                   
                   
                   
                 GCACTTCTCCTGGTTCGTGGGTGATCTGGAGGTGCAC 
                 (SEQ ID NO: 47) 
                   
                   
                   
               
               
                   
                   
                   
                 ACGGGCAGGTCGAAGCCGAGAGAGGAGCAGTTCAACA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCACCTACCGCGTGGTCAGCACCCTGCCCATCCAGCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAATGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTCAACAACAAAGGCCTCCCAGCCCCCATCGTGAGGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCATCTCCAGGACCAAAGGGCAGGCCCGCGAGCCGCA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGTGTACGTCCTGGCCCCACCCCAGGAAGAGCTCAGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAAAGCACGGTCAGCGTCACTTGCATGGTCACTGGCTT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTACCCAGACTACATCGCCGTAGAGTGGCATAGAGACC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGCAGGCTGAGTCGGAGGACAAGTACCGCACGACCCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCCCCAGCTGGACAGCGATGGCTCCTACTTCCTGTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGCAGGCTCAAGGTGAACAAGAACAGCTGGCAAGAAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GAGGCGCCTACACGTGTGTACTGATGCATGAGGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 48) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG3? 
                 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCAT 
                 ASTTAPKVYPLASSCGDTSSSTVTLGC 
                 NW_ 
                   
                   
               
               
                   
                   
                   
                 CCAGCTGCGGGGACACGTCCAGCTCCACCGTGACCCT 
                 LVSSYMPEPVTVTWNSGALKNGVHTF 
                 005784206 
                   
                   
               
               
                   
                   
                   
                 GGGCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTG 
                 PAVRQSSGLYSLSSMVTMPTSTAGTQ 
                   
                   
                   
               
               
                   
                   
                   
                 ACCGTGACCTGGAACTCGGGTGCCCTGAAGAACGGCG 
                 TFTCNVAHPASSTKVDTAVTARHPVP 
                   
                   
                   
               
               
                   
                   
                   
                 TGCACACCTTCCCGGCCGTCCGGCAGTCCTCCGGGCT 
                 KTPETPIHPVKPPTQEPRDEKTPCQCP 
                   
                   
                   
               
               
                   
                   
                   
                 CTACTCTCTCAGCAGCATGGTGAGCATGCCCACCAGGA 
                 KCPEPLGGLSVFIFPPKPKDTLTISGTP 
                   
                   
                   
               
               
                   
                   
                   
                 CCGCAGGAACCCAGACCTTCACCTGCAACGTAGCCCA 
                 EVTCVVVDVGQDDPEVQFSWFVDDVE 
                   
                   
                   
               
               
                   
                   
                   
                 CCCGGCCAGCAGCACCAAGGTGGACACGGCTGTCACT 
                 VHTARMKPREEQFNSTYRVVSALPIQH 
                   
                   
                   
               
               
                   
                   
                   
                 GCAAGGCATCCCGTCCCGAAGACACCAGAGACACCTA 
                 QDWLREKEFKCKVNNKGLPAPIVRTISR 
                   
                   
                   
               
               
                   
                   
                   
                 TCCATCCTTAAAACCCCCAACCCAGGAGCCCAGAGAT 
                 TKGQAREPQVYVLAPPREELSKSTLSL 
                   
                   
                   
               
               
                   
                   
                   
                 GAAAAGACACCCTGCCAGTGTCCCAAATGCCCAGAACC 
                 TCLITGFYPEEVDVEWQRNGQPESEDK 
                   
                   
                   
               
               
                   
                   
                   
                 TCTGGGAGGACTGTCTGTCTTCATCTTCCCACCGAAAC 
                 YHTTPPQLDADGSYFLYSRLRVNRSSW 
                   
                   
                   
               
               
                   
                   
                   
                 CCAAGGACACCCTCACAATCTCTGGAACGCCCGAGGT 
                 QEGDHYTCAVMHEALRNHYKEKPISRS 
                   
                   
                   
               
               
                   
                   
                   
                 CACGTGTGTGGTGGTGGACGTGGGCCAGGATGACCCC 
                 PGK* 
                   
                   
                   
               
               
                   
                   
                   
                 GAAGTGCAGTTCTCCTGGTTCGTGGATGACGTGGAGG 
                 (SEQ ID NO: 49) 
                   
                   
                   
               
               
                   
                   
                   
                 TGCAGACAGCCAGGATGAAGCCAAGAGAGGAGCAGTT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAACAGCACCTACCGCGTGGTCAGCGCCCTGCCCATC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CAGCACCAGGACTGGCTGCGGGAAAAGGAGTTCAAGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCAAGGTCAACAACAAAGGCCTCCCGGCCCCCATCGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GAGGACCATCTCCAGGACCAAAGGGCAGGCCCGGGAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCACAGGTGTATGTCCTGGCCCCACCCCGGGAAGAGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TCAGCAAAAGCACGCTCAGCCTCACCTGCCTAATCACC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGCTTCTACCCAGAAGAGGTAGACGTGGAGTGGCAGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GAAATGGGCAGCCTGAGTCAGAGGACAAGTACCACAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GACCCCACCCCAGCTGGACGCTGACGGCTCCTACTTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTGTACAGCAGGCTCAGGGTGAACAGGAGCAGCTGGC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGGAAGGAGACCACTACACGTGTCCAGTGATGCATGAA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCTTTACGGAATCACTACAAAGAGAAGCCCATCTCGAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTCTCCGGGTAAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 50) 
                   
                   
                   
                   
               
               
                   
                 Water 
                 Ig 
                 CAGCCCAAGTCCGCACCCTCAGTCACCCTGTTCCCAC 
                 QPKSAPSVTLFPPSTEELSANKATLVC 
                 NW_ 
                 Not 
                 None 
               
               
                   
                 buffalo Ig 
                 lambda? 
                 CCTCCACGGAGGAGCTCAGCGCCAACAAGGCCACCCT 
                 LISDFYPGSMTVARKADGSTITRNVETT 
                 0055690786 
                 registered 
                   
               
               
                   
                 light chain 
                   
                 GGTGTGTCTCATCAGCGACTTCTACCCGGGTAGCATGA 
                 RASKQSNSKYAASSYLSLTGSEWKSKG 
                   
                   
                   
               
               
                   
                 constant 
                   
                 CCGTGGCCAGGAAGGCAGACGGCAGCACCATCACCCG 
                 SYSCEVTHEGSTVTKTVKPSECS* 
                   
                   
                   
               
               
                   
                 region (CL) 
                   
                 GAACGTGGAGACCACCCGGGCCTCCAAACAGAGCAAC 
                 (SEQ ID NO: 51) 
                   
                   
                   
               
               
                   
                   
                   
                 AGCAAGTACGCCGCCAGCAGCTACCTGAGCCTGACGG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GCAGCGAGTGGAAATCGAAAGGCAGTTACAGCTGCGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GGTCACGCACGAGGGGAGCACCGTGACAAAGACAGTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAGCCCTCAGAGTGTTCTTAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 52) 
                   
                   
                   
                   
               
               
                   
               
               
                 Human 
                 Human Ig 
                 IgG4 
                 GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCA 
                 ESKYGPPCPSCPAPEFLGGPSVFLFPP 
                 K01316 
                 http://www. 
                 Ellison J. 
               
               
                 (Scientific 
                 heavy chain 
                 variant 
                 GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTG 
                 KPKDTLMISRTPEVTCVVVDVSQEDPE 
                   
                 IMGT 
                 et al., 
               
               
                 Name:  Homo   
                 constant 
                 1 
                 TTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC 
                 VQGNWYVDGVEVHNAKTKPREEQFNS 
                   
                 repertoire 
                 DNA, 1, 
               
               
                   sapiens ) 
                 region 
                   
                 CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACCTG 
                 TYRVVSVLTVLHQDWLNGKEYKCKVS 
                   
                 indes.php? 
                 11-18 
               
               
                   
                 (CH1 CH3) 
                   
                 AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC 
                 NKGLPSSIEKTISKAKGQPREPQVYTLP 
                   
                 section= 
                 (1981). 
               
               
                   
                   
                   
                 GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAG 
                 PSQEEMTKNQVSLTCLVKGFYPSDIAV 
                   
                 LocusGenes&amp; 
                 PMID: 
               
               
                   
                   
                   
                 CCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG 
                 EWESNGQPENNYKTTPPVLDSDGSFFL 
                   
                 repertoire= 
                 6299662 
               
               
                   
                   
                   
                 GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG 
                 YSRLTVDKSRWQEGNVFSCSVMHEAL 
                   
                 genetable&amp; 
                   
               
               
                   
                   
                   
                 AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA 
                 HNHYTQKSLSLSLGK* 
                   
                 species= 
                   
               
               
                   
                   
                   
                 GGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA 
                 (SEQ ID NO: 53) 
                   
                 human&amp;group= 
                   
               
               
                   
                   
                   
                 GCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC 
                   
                   
                 IGHC 
                   
               
               
                   
                   
                   
                 CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCCGAGAACAACTACAAGACCACGCCTCCCGTGCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 54) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG4 
                 GAGTCCAAATATCGTCCCCCGTGCCCATCATGCCCA 
                 ESKYGPPGPSCPAPEFLGGPSVFLFPP 
                 AJ001563 
                   
                   
               
               
                   
                   
                 variant 
                 GCACCTGAGTTCCTGGGCCGACCATCAGTCTTCCTG 
                 KPKDTLMISRTPEVTCVVVDVSQEDPE 
                   
                   
                   
               
               
                   
                   
                 2 
                 TTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC 
                 VQFNWYVDGVEVHNAKTKPREEQFNS 
                   
                   
                   
               
               
                   
                   
                   
                 CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG 
                 TRYVVSVLTVVHQDWLNGKEYKCKVS 
                   
                   
                   
               
               
                   
                   
                   
                 AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC 
                 NKGLPSSIEKTISKAKGQPREPQVYTLP 
                   
                   
                   
               
               
                   
                   
                   
                 GTGGATGCCGTGGAGGTGCATAATGCCAAGACAAAG 
                 PSQEEMTKNQVSLTCLVKGFYPSDIAV 
                   
                   
                   
               
               
                   
                   
                   
                 CCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG 
                 EWESNGQPENNYKTTPPVLDSDGSFFL 
                   
                   
                   
               
               
                   
                   
                   
                 GTCAGCGTCCTCACCGTCGTGCACCAGGACTGGCTG 
                 YSRLTVDKSRWQEGNVFSCSVMHEAL 
                   
                   
                   
               
               
                   
                   
                   
                 AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA 
                 NHNYTQKSLSLSLSGK* 
                   
                   
                   
               
               
                   
                   
                   
                 GGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA 
                 (SEQ ID NO: 55) 
                   
                   
                   
               
               
                   
                   
                   
                 GCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CACTACACGCAGAAGAGCCTCTCCCTGTCTCTGGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 56) 
                   
                   
                   
                   
               
               
                   
                   
                 IgG4 
                 GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTG 
                 APEFLGGPSVFLPPKPKDTLMISRTPE 
                 AJ001584 
                   
                   
               
               
                   
                   
                 variant 
                 TTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC 
                 VTCVVVDVSQEDPEVQFNWYVDGVEV 
                   
                   
                   
               
               
                   
                   
                 3 
                 CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG 
                 HNAKTKPREEQFNSTYRVVSLVTVLHQ 
                   
                   
                   
               
               
                   
                   
                   
                 AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC 
                 DWLNGKEYKCKVSNKGLPSSIEKTISKA 
                   
                   
                   
               
               
                   
                   
                   
                 GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAG 
                 KGQPREPQVYTLPPSQEEMTKNQVSL 
                   
                   
                   
               
               
                   
                   
                   
                 CCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG 
                 TCLVKGFYPSDIAVEWESNGQPENNYK 
                   
                   
                   
               
               
                   
                   
                   
                 GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG 
                 TTPPVLDSDGSFFLYSKLTVDKSRWQE 
                   
                   
                   
               
               
                   
                   
                   
                 AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA 
                 GNVFSCSVMHEALHNYHTQKSLSLSLG 
                   
                   
                   
               
               
                   
                   
                   
                 GGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA 
                 K* 
                   
                   
                   
               
               
                   
                   
                   
                 GCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC 
                 (SEQ ID NO: 57) 
                   
                   
                   
               
               
                   
                   
                   
                 CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AGCCACATCGCCGTCGAGTGGGAGAGCAATGGGCAG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG 
                   
                   
                   
                   
               
               
                   
                   
                   
                 GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 ACCGTGGACAAGAGCAGGTGGCAGGAGGGGAACGTC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC 
                   
                   
                   
                   
               
               
                   
                   
                   
                 CACTACACGCAGAAGAGCCTCTCCCTGTCTCTGGGT 
                   
                   
                   
                   
               
               
                   
                   
                   
                 AAATGA 
                   
                   
                   
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 58) 
                   
                   
                   
                   
               
               
                   
                 Human Ig 
                 Ig kappa 
                 ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA 
                 TVAAPSVFIFPPSDEQLKSGTASVVCL 
                 X96754 
                 http://www. 
                 None 
               
               
                   
                 light chain 
                 (CK) 
                 TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT 
                 LNNFYPREAKVQWKVDNALQSGNSQE 
                   
                 IMGT 
                   
               
               
                   
                 constant 
                   
                 GTGTGCCTGCTGAATAACTTCTATCCCAGAGACGCC 
                 SVTEQDSKDSTYSLSSTLTLSKADYEK 
                   
                 repertoire 
                   
               
               
                   
                 region 
                   
                 AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG 
                 HKVYACEVTHQGLSSPVTKSFNRGEC* 
                   
                 indes.php? 
                   
               
               
                   
                   
                   
                 GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGC 
                 (SEQ ID NO: 59) 
                   
                 section= 
                   
               
               
                   
                   
                   
                 AAGGACAGCACCTACAGCCTCAGCAGCACCCTGACG 
                   
                   
                 LocusGenes&amp; 
                   
               
               
                   
                   
                   
                 CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC 
                   
                   
                 repertoire= 
                   
               
               
                   
                   
                   
                 GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC 
                   
                   
                 genetable&amp; 
                   
               
               
                   
                   
                   
                 GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 
                   
                   
                 species= 
                   
               
               
                   
                   
                   
                 (SEQ ID NO: 60) 
                   
                   
                 human&amp;group= 
                   
               
               
                   
                   
                   
                   
                   
                   
                 IGHC 
               
               
                   
               
            
           
         
       
     
     The amino acid sequences as shown in SEQ ID NOS: 3, 21-28, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations are introduced, the resulting amino acid sequences may be capable of having the function as a constant region of Ig heavy chain or light chain. 
     Although the constant region of wild-type human IgG1 has ADCC activity and CDC activity, it is known that these activities can be reduced by introducing amino acid substitutions and deletions into specific sites. In the case of animals other than human where the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, mutations may be introduced into the relevant region of an immunoglobulin equivalent to human IgG1 so that the resultant constant region with reduced ADCC activity and CDC activity can be used. 
     The present invention provides an artificial genetic DNA comprising (a′) a DNA encoding a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat and (b′) a DNA encoding a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 19) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat. The present invention also provides a DNA encoding a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat. Further, the present invention also provides a DNA encoding a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 19) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat. 
     For (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLEYSDGYTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of GVS and CDR3 having the amino acid sequence of FQATHDPDT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFSLTSYY (SEQ ID NO: 18), CDR2 having the amino acid sequence of IRSGGST (SEQ ID NO: 19) and CDR3 having the amino acid sequence of ARTSSGYEGGFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat, reference should be had to the foregoing description. The DNA of (a′) is a DNA (gene) encoding the light chain of (a); and the DNA of (b′) is a DNA (gene) encoding the heavy chain of (b). An artificial genetic DNA comprising the DNA of (a′) and the DNA of (′b) may be synthesized on commercial synthesizer. Restriction enzyme recognition sites, KOZAK sequences, poly-A addition signal sequences, promoter sequences, intron sequences or the like may be added to the artificial genetic DNA. 
     The present invention also provides a vector comprising the above-mentioned artificial genetic DNA. 
     As the vector,  Escherichia coli -derived plasmids (e.g., pBR322, pBR325, pUC12 or pUC13):  Bacillus subtilis -derived plasmids (e.g., pUB110, pTP5 or pC194), yeast-derived plasmids e.g., pSH19 or pSH15); bacteriophages such as λ phage; animal viruses such as retrovirus or vaccinia virus; or insect pathogen viruses such as baculovirus may be used. In the Examples described later, pDN112 (Marti A, Yoshida R, Miyamoto H, Ishijima M, Suzuki Y, Higuchi M, Matsuyama Y, Igarashi M, Nakayama E, Kuroda M, Saijo M, Feldmann F, Brining D, Feldmann H, Takada A. PLoS One, 7: e36192, Apr. 27, 2012; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology, 142(4): 551-561, Aug. 2014) was used. 
     The vector may also comprise promoters, enhancers, splicing signals, poly-A addition signals, intron sequences, selection markers, SV40 replication origins, and so forth. 
     The present invention also provides a host cell transformed by the above vector. It is possible to prepare the anti-PD-1 antibody of the invention by culturing the host cell and collecting the antibody of interest from the resultant culture. Therefore, the present invention also provides a method of preparing an antibody, comprising culturing the above-described host cell and collecting the anti-PD-1 antibody of the invention from the culture. In the method of the present invention for preparing an antibody, a vector incorporating an artificial genetic DNA comprising a DNA encoding the light chain and a DNA encoding the heavy chain may be transfected into a host cell. Alternatively, a vector incorporating a DNA encoding the light chain and a vector incorporating a DNA encoding the heavy chain may be co-transfected into a host cell. 
     Examples of the host cell include, but are not limited to, bacterial cells (such as  Escherichia bacteria, Bacillus  bacteria or  Bacillus subtilis ), fungal cells (such as yeast or  Aspergillus ), insect cells (such as S2 cells or Sf cells), animal cells (such as CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells or HEK 293 cells) and plant cells. Among these, CHO-DG44 cell (CHO-DG44(dfhr -/- )) which is a dihydrofolate reductase deficient cell is preferable. 
     Introduction of a recombinant vector into a host cell may be performed by the methods disclosed in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (e.g., the calcium phosphate method, the DEAE-dextran method, transfection, microinjection, lipofection, electroporation, transduction, scrape loading, the shotgun method, etc.) or by infection. 
     The resultant transformant may be cultured in a medium, followed by collection of the anti-PD-1 antibody of the present invention from the culture. When the antibody is secreted into the medium, the medium may be recovered, followed by isolation and purification of the antibody from the medium. When the antibody is produced within the transformed cells, the cells may be lysed, followed by isolation and purification of the antibody from the cell lysate. 
     Examples of the medium include, but are not limited to, OptiCHO medium, Dynamis medium, CD CHO medium, ActiCHO medium, FortiCHO medium, Ex-Cell CD CHO medium, BalanCD CHO medium, ProCHO 5 medium and Cellvento CHO-100 medium. 
     The pH of the medium varies depending on the cell to be cultured. Generally, a pH range from 6,8 to 7.6 is used; mostly, a pH range from 7.0 to 7.4 is appropriate. 
     When the cell to be cultured is CHO cells, culture may be performed by methods known to those skilled in the art. For example, it is usually possible to perform culturing in a gas-phase atmosphere having a CO 2  concentration of 0-40%, preferably 2-10%, at 30-39° C., preferably around 37° C. 
     The appropriate period of culture is usually from one day to three months, preferably from one day to three weeks. 
     Isolation and purification of the antibody may be performed by known methods. Known isolation/purification methods which may be used in the present invention include, but are not limited to, methods using difference in solubility (such as salting-out or solvent precipitation); methods using difference in molecular weight (such as dialysis, ultrafiltration, gel filtration or SDS-polyactylamide gel electrophoresis); methods using difference in electric charge (such as ion exchange chromatography); methods using specific affinity (such as affinity chromatography); methods using difference in hydrophobicity (such as reversed phase high performance liquid chromatography); and methods using difference in isoelectric point (such as isoelectric focusing). 
     The anti-PD-1 antibody of the present invention may be used as an antibody drug for animals or human. Therefore, the present invention provides a pharmaceutical composition comprising the above-described anti-PD-1 antibody as an active ingredient. 
     The pharmaceutical composition of the present invention may be used for prevention and/or treatment of cancers and/or infections. Examples of cancers and/or infections include, but are not limited to, neoplastic diseases (e.g., malignant melanoma, lung cancer, gastric cancer, renal cancer, breast cancer, bladder cancer, esophageal cancer, ovarian cancer and the like), leukemia, Johne&#39;s disease, anaplasmosis, bacterial mastitis, mycotic mastitis, mycoplasma infections (such as mycoplasma mastitis, mycoplasma pneumonia or the like), tuberculosis,  Theileria orientalis  infection, cryptosporidiosis, coccidiosis, trypanosomiasis and leishmaniasis. 
     The anti-PD-1 antibody of the present invention may be dissolved in buffers such as PBS, physiological saline or sterile water, optionally filter-sterilized with a filter or the like and then administered to animal subjects (including human) by injection. To the solution of this antibody, additives (such as coloring agents, emulsifiers, suspending agents, surfactants, solubilizers, stabilizers, preservatives, antioxidants, buffers, isotonizing agents, pH adjusters and the like) may be added. As routes of administration, intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal administration and the like may be selected. Transnasal or oral administration may also be used. 
     The dose and the number of times and frequency of administration of the anti-PD-1 antibody of the present invention may vary depending on the symptoms, age and body weight of the animal subject, the method of administration, the dosage form and so on. For example, 0.1-100 mg/kg body weight, preferably 1-10 mg/kg body weight, per adult animal may usually be administered at least once at such a frequency that enables confirmation of desired effect. 
     While the pharmaceutical composition of the present invention may be used alone, it may be used in combination with surgical operations, radiation therapies, other immunotherapies such as cancer vaccine, or molecular target drugs. Synergistic effect can be expected from such combinations. 
     EXAMPLES 
     Hereinbelow, the present invention will be described in more detail with reference to the following Examples. However, the present invention is not limited to these Examples. 
     Example 1 
     Establishment of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody Introduction 
     Programmed cell death 1 (PD-1), an immunoinhibitory receptor, and its ligand programmed cell death ligand 1 (PD-L1) are molecules identified by Prof, Tasuku Honjo et al., Kyoto University, as factors which inhibit excessive immune response and are deeply involved in immunotolerance. Recently, it has been elucidated that these molecules are also involved in immunosuppression in tumors. In the subject Example, for the purpose of establishing a novel therapy for bovine infections, a chimeric antibody gene was prepared in which variable region genes of rat anti-bovine PD-1 monoclonal antibody 5D2 capable of inhibiting the binding of bovine PD-1 to PD-L1 were linked to constant region genes of bovine immunoglobulins (bovine IgG1 and Igλ, with mutations having been introduced into the putative binding sites of Fcγ receptors in bovine IgG1&#39;s CH2 domain to inhibit ADCC activity; see  FIGS. 1 and 11  for amino acid numbers and mutations: 250 E→P, 251 L→V, 252 P→A, 253 G→deletion, 347 A→S, 348 P→S; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S. Ohashi K. Immunology, 142(4): 551-561, Aug. 2014). The resultant chimeric antibody gene was introduced into Chinese hamster ovary cells (DHO cells), which were cultured and proliferated to produce a rat-bovine chimeric anti-bovine PD-1antibody ch5D2. The effect of this chimeric antibody was confirmed in vitro and in vivo. 
     Materials, Methods and Experimental Results 
     2.1. Construction of Bovine PD-1 and PD-L1 Expressing Cells 
     The nucleotide sequences of the full length cDNAs of bovine PD-1 gene (GenBank accession number AB510901; Ikebuchi R, Konnai 5, Sunden Y, Onuma M, Ohashi K. Microbiol. Immunol., 54(5): 291-298; May 2010) and bovine PD-Li gene (GenBank accession number AB510902; Ikebuchi R, Konnai S, Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K. Vet. Res., 42: 103, Sep. 26. 2011) were determined. Based on the resultant genetic information, bovine PD-1 and bovine PD-L1 expressing cells were prepared. First, for preparing bovine PD-1 or PD-L1 expressing plasmid, PCR was performed using a synthesized bovine peripheral blood mononuclear cell (PBMC)-derived cDNA as a template and designed primers having NotI and HindIII (bovine PD-1) recognition sites or NheI and XhoI (bovine PD-L1) recognition sites on the 5′ side (boPD-1-myc F and R; or boPD-L1-EGFP F and R). The PCR products were digested with Nod (Takara) and HindIII (Takara; bovine PD-1) or NheI (Takara) and XhoI (Takara; bovine PD-L1), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCMV-Tag1 vector (Agilent Technologies; bovine PD-1) or pEGFP-N2 vector (Clontech; bovine PD-L1) treated with the restriction enzymes in the same manner. The resultant expression plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCMV- 
     
       
         
           
               
               
            
               
                   
                 Tag1-boPD-1 or pEGFP-N2-boPD-L1. 
               
               
                   
                 Primer (boPD-1-myc F): 
               
               
                   
                 (SEQ ID NO: 61) 
               
               
                   
                 ATATGCGGCCGCATGGGGACCCCGCGGGCGCT 
               
               
                   
                   
               
               
                   
                 Primer (boPD-1-myc R): 
               
               
                   
                 (SEQ ID NO: 62) 
               
               
                   
                 GCGCAAGCTTTCAGAGGGGCCAGGAGCAGT 
               
               
                   
                   
               
               
                   
                 Primer (boPD-L1-EGFP F): 
               
               
                   
                 (SEQ ID NO: 63) 
               
               
                   
                 CTAGCTAGCACCATGAGGATATATAGTGTCTTAAC 
               
               
                   
                   
               
               
                   
                 Primer (boPD-L1-EGFP R): 
               
               
                   
                 (SEQ ID NO: 64) 
               
               
                   
                 CAATCTCGAGTTACAGACAGAAGATGACTGC 
               
            
           
         
       
     
     Bovine PD-1 expressing cells were prepared by the procedures described below. First, 2.5 μg of pCMV-Tag1-boPD-1 was introduced into 4×10 6  CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science), 20 ml/L GlutaMAX supplement (Life Technologies), and 18 ml/L 10% Pluronic F-68 (Life Technologies), followed by selection. The resultant expression cells were reacted with rat anti-bovine PD-1 antibody 5D2 at room temperature. After washing, the cells were further reacted with anti-rat IgG microbead-labeled antibody (Miltenyi Biotec) at room temperature. Cells expressing bovine PD-1 at high levels were isolated with Auto MACS (Miltenyi Biotec). Subsequently, re-isolation was performed in the same manner to obtain still higher purity. The resultant expression cells were subjected to cloning by limiting dilution to thereby obtain a CHO DG44 cell clone expressing bovine PD-1 at high level (bovine PD-1 expressing cells). 
     Bovine PD-L1 membrane expressing cells were prepared by the procedures described below. First, 2.5 μg of pEGFP-N2-boPD-L1 or pEGFP-N2 (negative control) was introduced into 4×10 6  CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science), 20 ml/L GlutaMAX supplement (Life Technologies) and 18 ml/L 10% Pluronic F-68 (Life Technologies), followed by selection and cloning by limiting dilution (bovine PD-L1 expressing cell clone). In order to confirm the expression of bovine PD-L1 in the thus prepared cell clone, intracellular localization of EGFP was visualized with an inverted confocal laser microscope LSM700 (ZEISS). 
     2.2 Construction of Soluble Bovine PD-1 
     Bovine PD-1-Ig expressing plasmid was constructed by the procedures described below. Briefly, the signal peptide and the extracellular region of bovine PD-1 (GenBank accession number AB510901) were linked to the constant region of a known bovine IgG1 (GenBank accession number X62916) to prepare a gene sequence. After codons were optimized for CHO cells, gene synthesis was performed in such a manner that NotI recognition sequence, KOZAK sequence, bovine PD-1 signal peptide sequence, bovine PD-1 gene extracellular region sequence, bovine IgG1 Fc region sequence, and XbaI recognition sequence would be located in the gene in this order. It should be noted here that bovine IgG1 was mutated to inhibit ADCC activity; more specifically, mutations were introduced into the putative binding sites for Fcγ receptors of CH2 domain s of mutation: 185 E→P, 186 L→V, 187 P→A, 189 G→deletion, 281 A→S, 282 P→S; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y; Murata S, Ohashi K. Immunology, 142(4): 551-561, Aug. 204; the amino acid sequence of PD-1-Ig and the sites of mutation are disclosed in  FIG. 2  of this article). The synthesized gene strand was digested with NotI (Takara) and XbaI (Takata), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics), and incorporated into the cloning site (NotI and XbaI restriction enzyme recognition sequences downstream of PCMV and between INRBG and PABGH) of expression vector pDN11 (kindly provided by Prof. S. Suzuki, Hokkaido University Research Center for Zoonosis Control) treated with the restriction enzymes in the same manner, whereby bovine PD-1-Ig expressing vector was constructed. The expression plasmid was purified with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pDN11-boPD-1-Ig. 
     Bovine PD-1-His expressing plasmid was prepared by the procedures described below. Briefly, for the purpose of amplifying the signal peptide and the extracellular region of bovine PD-1 (GenBank accession number AB510901), primers were designed in which NotI and XhoI recognition sites were added on the 5′ side (boPD-1-His F and R). A genetic sequence encoding a 6×His tag was added to the reverse primer. PCR was performed using a synthesized bovine PBMC-derived cDNA as a template. The respective PCR products were digested with NotI (Takara) and XhoI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCXN2.1(+) vector (Niwa H, Yamamura K, Miyazaki J. Gene, 108(2): 193-199; Dec. 15, 1991; kindly provided by Dr. T. Yokomizo, Juntendo University Graduate School of Medicine) treated with the restriction enzymes in the same manner. The resultant expression plasmid was purified with FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCXN2.1-boPD-1-His. 
     
       
         
           
               
            
               
                 Primer (boPD-1-His F): 
               
               
                 (SEQ ID NO: 65) 
               
               
                 ATAAGAATGCGGCCGCCACCATGGGGACCCCGCGGGCGCT  
               
               
                   
               
               
                 Primer (boPD-1-His R): 
               
               
                 (SEQ ID NO: 66) 
               
               
                 GCCCTCGAGTTATGGTGATGGTGATGGTGGATGACCAGGCTCTGCATCT 
               
            
           
         
       
     
     Soluble bovine PD-1-Ig expressing cells were prepared by the procedures described below. Briefly, 2.5 μg of pDN11-boPD-1-Ig was introduced into 4×10 6  CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD OptiCHO medium (Life Technologies) containing 800 μg/ml G418 (Enzo Life Science) and 20 ml/L GlutaMAX supplement (Life Technologies). After cultured for 3 weeks, the cells were subjected to selection. Briefly, the concentrations of the Fc fusion recombinant protein in the culture supernatants of the resultant cell clones were measured by ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to thereby select those cell clones that express the Fc fusion recombinant protein at high levels. The resultant highly expressing cell clone was transferred to a G418-free medium and cultured under shaking for 14 days, followed by collection of a culture supernatant. The culture supernatant containing the Fc fusion recombinants protein was ultrafiltered with Centricon Plus-70 (Millipore). Then, the Fc fusion recombinant protein was purified with Ab-Capcher Extra (ProteNova). After purification, the buffer was exchanged with phosphate-buffered physiological saline (PBS; pH 7.4) using PD-10 Desalting Column (GE Healthcare). The resultant protein was stored at −30° C. until use in experiments (bovine PD-1-Ig), The concentration of the purified bovine PD-1-1g was measured by ELISA using IgG F(c) rabbit polyclonal antibody (Rockland). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric). 
     Soluble bovine PD-1-His expressing cells were prepared by the procedures described below. Briefly, 30 μg of pCXN2.1-boPD-1-His was introduced into 7.5×10 7  Expi293F cells (Life Technologies) using Expifectamine (Life Technologies). After a 7-day culture under shaking, the culture supernatant was collected. The recombinant protein of interest was purified from the culture supernatant using TALON Metal Affinity Resin (Clontech; bovine PD-1-His). After purification, the buffer was exchanged with PBS (pH 7.4) using PD MiniTrap G-25 (GE Healthcare). The resultant protein was stored at −30° C. until use in experiments (bovine PD-1-His), The concentration of purified bovine PD-1-His was quantitatively determined in terms of the absorbance (280 nm) measured with Nanodrop8000 Spectrophotometer (Thermo Fisher Scientific). 
     2.3. Preparation of Rat Anti-Bovine PD-1 Monoclonal Antibody Producing Cells 
     Rat was immunized in the footpad with bovine PD-1-Ig (described above). Hybridomas were established by the iliac lymph node method to thereby obtain rat anti-bovine PD-1 monoclonal antibody producing hybridoma 5D2. With respect to the method of establishment of rat anti-bovine PD-1 monoclonal antibody, details are disclosed in the following non-patent document (Ikebuchi R, Konnai S, Okagawa I, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Vet. Res. 44: 59; Jul. 22, 2013). 
     2.4. Preparation of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody Expressing Vector 
     Rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was established by fusing the antibody constant regions of bovine IgG1 and Igλ, with rat anti-bovine PD-1 antibody 5D2 being used as antibody variable regions. 
     First, the genes of heavy chain and light chain variable regions were identified by the RACE method from a hybridoma that would produce rat anti-bovine PD-1 antibody 5D2. Subsequently, a gene sequence was prepared in which the heavy chain and the light chain variable regions of the rat anti-bovine PD-1 antibody 5D2 were linked to known constant regions of bovine IgG1 (heavy chain, modified from GenBank Accession number X62916) and bovine Ig2 (light chain; GenBank Accession number X62917), respectively. Then, codon optimization was carried out (SEQ ID NOS: 9 and 10 (amino acid sequences); SEQ ID NOS: 14 and 15 (nucleotide sequences after codon optimization)). It should be noted that bovine IgG1 had mutations added to the putative binding sites of Fcγ receptors in CH2 domain in order to suppress ADCC activity (See  FIGS. 1 and 11  for amino acid numbers and mutations: 251 E→P, 252 L→V, 253 P→A, 254 G→deletion, 348 A→S, 349 P→S; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology, 142(4): 551-561; Aug. 2014). Then, the gene was artificially synthesized in such a manner that NotI recognition sequence, KOZAK sequence, chimeric antibody light chain sequence, poly-A addition signal sequence (PABGH), promoter sequence (PCMV), SacI recognition sequence, intron sequence (INRBG), KOZAK sequence, chimeric antibody heavy chain sequence and XbaI recognition sequence would be located in this order. The synthesized gene strand was digested with NotI (Takara) and XbaI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into the cloning site (NotI and XbaI restriction enzyme recognition sequences downstream of PCMV and between INRBG and PABGH) of expression plasmid pDN112 (kindly provided by Prof. S. Suzuki, Hokkaido University Research Center for Zoonosis Control) treated with the restriction enzymes in the same manner ( FIG. 2 ). The resultant plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pDN112-boPD-1ch5D2. 
     2.5. Expression of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody (FIG.  3 ) 
     The pDN112-boPD-1ch5D2 prepared above was transfected into CHO-DG44 cells (CHO-DG44(dfhr -/- )) which were a dihydrofolate reductase deficient cell. Forty-eight hours later, the medium was exchanged with CD OptiCHO medium (Life Technologies) containing 2 mM GlutaMAX supplement (Life Technologies) and 800 μg/ml G418 sulfate (Enzo Life Science). After cultured for 3 weeks, the expression cells were subjected to selection and cloning by limiting dilution. Subsequently, the concentrations of the chimeric antibody in the culture supernatants were measured by dot blotting and ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to thereby select high expression clones. Further, to the selected clones expressing rat-bovine chimeric anti-bovine PD-1 antibody at high levels were subjected to gene amplification treatment by adding a load with 60 nM methotrexate (Mtx; Wako)-containing medium. The thus established cell clone stably expressing rat-bovine chimeric anti-bovine PD-1 antibody was transferred into Mtx-free CD Opti-CHO medium and cultured wider shaking for 14 days (125 rpm, 37° C., 5% CO 2 ). Chimeric antibody production in the culture supernatant was measured by ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric). The culture supernatant at day 14 was centrifuged at 10,000 g for 10 min to remove cells, and the centrifugal supernatant was passed through a Steritop-GP 0.22 μm filter (Millipore) for sterilization and then stored at 4° C. until it was subjected to purification. 
     The results are shown in  FIG. 3A . Among the rat-bovine chimeric anti-bovine PD-1 antibody expressing cell clones, the most productive clone secreted 91.7 mg/l of the chimeric antibody into the culture supernatant during the 14-day culture under shaking. 
     2.6. Purification of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody 
     From the culture supernatant prepared as described above, each chimeric antibody was purified using Ab Capcher Extra (ProteNova). An open column method was used for binding to resin; 1.5 M Glycine/3 M NaCl (pH 8.0) was used as equilibration buffer and wash buffer. As elution buffer, 0.1 M Glycine-HCl (pH 2.8) was used. As neutralization buffer, 1 M Tris (pH 9.0) was used. The purified antibody was subjected to buffer exchange with PBS (pH 7.41) using PD-10 Desalting Column (GE Healthcare) and concentrated using Amicon Ultra-1.5 (50 kDa, Millipore). The thus purified chimeric antibody was passed through a 0.22 μm syringe filter (Pall Life Sciences) for sterilization and stored at 4° C. until use in experiments. 
     2.7. Confirmation of the Purity of Purified Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody (FIG.  3 ) 
     In order to confirm the purity of purified rat-bovine chimeric anti-bovine PD-1 antibody, antibody proteins were detected by SDS-PAGE and CBB staining. Purified rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was suspended in Laemmli Sample Buffer (Bio-Rad) and denatured at 95° C. for 5 min under reducing conditions (reduced with 2-mercaptoethaanol; Sigma-Aldrich) or under non-reducing conditions. The thus prepared samples were electrophoresed using 10% polyacrylamide gel. As molecular weight markers, Precision Plus Protein All Blue Standards (Bio-Rad) were used. After electrophoresis, the gel was stained with Quick-CBB kit (Wako) and subsequently decolored in distilled water. 
     The results are shown in  FIG. 3B . Bands of rat-bovine chimeric anti-bovine PD-1 antibody were observed at predicted positions, that is, at 25 kDa (light chain) and 50 kDa (heavy chain) under reducing conditions and at 150 kDa under non-reducing conditions. 
     2.8. Binding Specificity of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody (FIG.  4 ) 
     It was confirmed by flow cytometry that rat-bovine chimeric anti-bovine PD-1 antibody specifically binds to bovine PD-1 expressing cells (described above). First, rat anti-bovine PD-1 antibody 5D2 or rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was reacted with bovine PD-1 expressing cells at room temperature for 30 min. After washing, Allophycocyanine (APC)-labeled anti-rat Ig goat antibody (Southern Biotech) or Alexa Fluor 647-labeled anti-bovine IgG (H+L) goat F(ab′)2 (Jackson ImmunoResearch) was reacted at room temperature for 30 min. As negative control antibody, rat IgG2a (κ) isotype control (BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After washing, each rat antibody or rat-bovine chimeric antibody bound to cell surfaces was detected by FACS Verse (BD Biosciences). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. 
     The experimental results are shown in  FIG. 4 . It was revealed that rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 binds to bovine PD-1 expressing cells in the same manner as rat anti-bovine PD-1 antibody 5D2. 
     2.9. PD-1 Binding Avidity of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody 
     The binding avidities to bovine PD-1 of rat anti-bovine PD-1 antibody 5D2 and rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 were measured by surface plasmon resonance using a bimolecular interaction analyzer (Biacore X100). Briefly, bovine PD-1-His (described above) was immobilized on a CM5 sensor chip (GE Healthcare) as a ligand. Subsequently, rat anti-bovine PD-1 antibody 5D2 or rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 was reacted as an analyte, followed by single kinetics analysis. The experiment was repeated 3 times under the same conditions. Binding constant (kd value) and dissociation constant (ka value) were determined in each experiment, and binding avidity (KD value) was obtained. 
     The experimental results are shown in the table below. The binding avidity of rat-bovine chimeric anti-bovine PD-1 antibody for PD-1 protein was similar to that of rat anti-bovine PD-1 antibody 5D2, with no statistical difference observed (p&gt;005; Welch&#39;s t-test). 
     
       
         
           
               
               
               
               
             
               
                   
               
               
                 Anti- 
                   
                   
                   
               
               
                 Bovine 
               
               
                 PD-1 
               
               
                 Antibody 
                 ka (1/Ms) 
                 kd (1/s) 
                 KD (M) 
               
               
                   
               
             
            
               
                 5D2 
                 1.84 × 10 4  ± 0.27 
                 2.15 × 10 −4  ± 0.44 
                 1.22 × 10 −8  ± 0.39 
               
               
                 ch5D2 
                 2.07 × 10 4  ± 0.06 
                 2.16 × 10 −4  ± 1.12 
                 1.05 × 10 −8  ± 0.58 
               
               
                   
               
            
           
         
       
     
     2.10. Blockade of Bovine PD-1/PD-L1 Binding by Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody (FIG.  5 ) 
     Using bovine PD-L1 expressing cells (described above) and bovine PD-1-Ig (described above), bovine PD-1/PD-L1 binding inhibition by anti-PD-1 antibodies was tested. First, rat anti-bovine PD-1 antibody 5D2 or rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 (final concentration: 0, 0,39, 0.78, 1.56, 3.12, 625, 12.5, 25 or 50 μg/ml) and bovine PD-1-Ig (final concentration: 5 μg/ml) labeled with biotin using Lightning-Link Type A Biotin Labeling Kit (Innova Biosciences) were added to 96-well plates, followed by reaction at 37° C. for 30 min. The resultant mixture was reacted with 1×10 5  bovine PD-L1 expressing cells at 37° C. for 30 min. After washing, the reaction mixture was reacted with APC-labeled streptavidin (BioLegend) at room temperature for 30 min to thereby detect bovine PD-1-Ig bound to cell surfaces. For analysis. FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. Taking the proportion of bovine PD-1-Ig-bound cells without addition of antibodies as 100%, the proportion of bovine PD-1-Ig-bound cells at each antibody concentration was shown as a relative value. 
     The experimental results are shown in  FIG. 5 . Rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 inhibited the binding of PD-1-Ig to PD-L1 expressing cells by the same degree as rat anti-bovine PD-1 antibody 5D2 did. 
     2.11. CDR Analysis of Rat Anti-Bovine PD-1 Antibody 
     The complementarity-determining regions (CDRs) of rat anti-bovine PD-1 antibody 5D2 were determined using NCBI IGBLAST (http://www.ncbi.nlm.nih.gov/igblast/). The results are shown in  FIG. 1 . 
     2.12. Inoculation Test on Cattle with Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody 
     Established rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 (14 mg; 0.08 mg/kg) was intravenously administrated into an experimentally BIN-infected calf (Holstein, male, 4 months old, 173.5 kg). Blood samples were collected chronologically from the infected calf, followed by collection of blood (with heparin sodium (Ajinomoto) used as anticoagulant) and serum. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation using Percoll (GE Healthcare). 
     2.13. Kinetics of Administered Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody in Blood (FIG.  6 ) 
     Bovine PD-1-His (described above) was immobilized on ELISA plates (H type, Sumitomo Bakelite) at a final concentration of 10 μg/ml at 4° C. overnight. Subsequently, each well was washed with 200 μl of 0.05% Tween 20-supplemented Tris-buffered saline (TBS-T) five times, followed by blocking with 1% skim milk-supplemented TBS-T at room temperature for 1 hr. Another washing was carried out in the same manner. The serum collected from the test calf was added to each well and reacted at room temperature for 1 hr. After washing, horseradish peroxidase-labeled anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) was reacted at room temperature for 1 hr. Each well was washed again and then TMB One Component Substrate (Bethyl) was added for coloring. The enzyme reaction was terminated with 0.18 M dilute sulfuric acid. Absorbance (450 nm) was measured with Microplate Reader MTP-650FA (Corona Electric). For every plate washing operation, Auto Plate Washer BIO WASHER 50 (DS Pharma. Biomedical) was used. 
     The experimental results are shown in  FIG. 6 . Rat-bovine chimeric anti-bovine PD-1 antibody was detected in the serum of the test calf until 70 days after administration (at the end of the clinical test). The antibody retained particularly high concentrations for one week after administration. 
     2.14. Cell Proliferation Response of T Cells to BIN Antigen (FIG.  7 ) 
     Bovine PBMCs were suspended in PBS and reacted with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) at room temperature for 20 min for labeling. After washing twice with RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), penicillin 200 U/ml, streptomycin 200 μg/ml and 0.01% L-glutamine (Life Technologies), cell concentration was adjusted to 4×10 5  cells/ml using the same medium. To the PBMCs, culture supernatant of 2% BLV-infected fetal lamb kidney cell (FLK-BLV), culture supernatant of fetal lamb kidney cell (FLK) not infected with 2% BLV, or BLV gp51 peptide mix 0.1 μg/ml or 1 μg/ml was added, followed by a 6-day culture at 37° C. under 5% CO 2 . After 6 days, PBMCs were recovered and reacted with Alexa Fluor 647-labeled mouse anti-bovine CD4 antibody (CC30, AbD Serotec). Peridinin-chlorophyll-protein complex/cyanin 5.5-labeled mouse anti-bovine CD8 antibody (CC63, AbD Serotec) and R-Phycoerythrin/cyanin 7 (PE/Cy7)-labeled anti-bovine IgM mouse antibody (IL-A30, AbD Serotec) at 4° C. for 20 min. For labeling antibodies, Zenon Mouse IgG1 Labeling Kits (Life Technologies) or Lightning-Link Kits (Innova Biosciences) was used. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. With respect to the proportion of proliferated T cells (CFSE low  cells), statistical test was performed using the method of Dunnett. 
     The experimental results are shown in  FIG. 7 . Upon administration of rat-bovine chimeric anti-bovine PD-1 antibody, BLV-specific cell proliferative response in CD4 +  T cells showed a statistically significant increase immediately after the administration, compared to the response before administration. 
     2.15. Transition in BLV Proviral Load (FIG.  8 ) 
     DNA was extracted from isolated bovine PBMCs using Wizard DNA Purification kit (Promega). The concentration of the extracted DNA was quantitatively determined based on the absorbance (260 nm) measured with Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific). For measuring BIN proviral load in PBMCs, real time PCR was performed using Cycleave PCR Reaction Mix SP (Takara) and Probe/Primer/Positive control (Takara) for bovine leukemia virus detection. LightCycler480 System II (Roche Diagnosis) was used for the measurement. With respect to the measured proviral load, statistical test was performed by the method of Dunnett. 
     The experimental results are shown in  FIG. 8 . Upon administration of rat-bovine chimeric anti-bovine PD-1 antibody, BLV proviral load in PBMCs showed a statistically significant decrease immediately after the administration, compared to the load before administration. The BLV proviral load remained at low levels until the end of the clinical test (day 70). 
     Example 2 
     Application of Anti-PD-1 Antibody to Other Animal Species 
     1. Materials, Methods and Experimental Results 
     1.1. Identification of Ovine and Water Buffalo PD-1 Genes 
     In order to determine the full-lengths of the coding sequences (CDSs) of ovine and water buffalo PD-1 cDNAs, primers for amplifying the full lengths of CDSs were first designed (ovPD-1 CDS F and R; buPD-1 CDS F1, R1, F2 and R2) based on the nucleotide sequences of ovine and water buffalo PD-1 genes (GenBank accession numbers BC123854 and XM 012176227), and then PCR was performed using a synthesized ovine or water buffalo PBMC-derived cDNA as a template. For the resultant amplified products, nucleotide sequences were determined with a capillary sequencer according to conventional methods (Mingala C N, Konnai S, Ikebuchi R, Ohashi K. Comp. Immunol. Microbiol. Infect. Dis., 34(1): 55-63; Jan. 2011; Water buffalo PD-1 gene was identified in this article). 
     
       
         
           
               
               
            
               
                   
                 Primer (ovPD-1 CDS F): 
               
               
                   
                 (SEQ ID NO: 67) 
               
               
                   
                 ATGGGGACCCCGCGGGCGCC 
               
               
                   
                   
               
               
                   
                 Primer (ovPD-1 CDS R): 
               
               
                   
                 (SEQ ID NO: 68) 
               
               
                   
                 TCAGAGGGGCCAGGAGCAGTGTCCA 
               
               
                   
                   
               
               
                   
                 Primer (buPD-1 CDS F1): 
               
               
                   
                 (SEQ ID NO: 69) 
               
               
                   
                 ATGGGGACCCCGCGGGCGCT 
               
               
                   
                   
               
               
                   
                 Primer (buPD-1 CDS R1): 
               
               
                   
                 (SEQ ID NO: 70) 
               
               
                   
                 GATGACCAGGCTCTGCATCT 
               
               
                   
                   
               
               
                   
                 Primer (buPD-1 CDS F2): 
               
               
                   
                 (SEQ ID NO: 71) 
               
               
                   
                 AATGACAGCGGCGTCTACTT 
               
               
                   
                   
               
               
                   
                 Primer (buPD-1 CDS R2): 
               
               
                   
                 (SEQ ID NO: 72) 
               
               
                   
                 TCAGAGGGGCCAGGAGCAGT 
               
            
           
         
       
     
     1.2. Construction of Ovine PD-1 Expressing COS-7 Cells 
     In order to prepare ovine PD-1 expression plasmid, PCR was performed using a synthesized ovine PBMC-derived cDNA as a template and primers designed by adding BglII and SmaI recognition sites on the 5′ side (ovPD-1-EGFP F and R). The resultant PCR products were digested with BglII (Takara) and SmaI (Takara), purified with FastGene Gel/PCR. Extraction Kit (NIPPON Genetics) and cloned into pEGFP-N2 vector (Clontech) treated with the restriction enzymes in the same manner. The expression plasmid of interest was extracted using FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C., until use in experiments. Hereinafter, the thus prepared plasmid is designated as pEGFP-N2-ovPD-1. 
     
       
         
           
               
               
            
               
                   
                 Primer (ovPD-1-EGFP F): 
               
               
                   
                 (SEQ ID NO: 73) 
               
               
                   
                 GAAGATCTATGGGGACCCCGCGGGCGCCG 
               
               
                   
                   
               
               
                   
                 Primer (ovPD-1-EGFP R): 
               
               
                   
                 (SEQ ID NO: 74) 
               
               
                   
                 GACCCGGGGAGGGGCCAGGAGCAGTGTCC 
               
            
           
         
       
     
     COS-7 cells were subcultured at a density of 5×10 4  cells/cm 2  in 6-well plates, and then cultured overnight in RPMI 1640 medium containing 10% inactivated fetal bovine serum (Invitrogen) and 0.01% L-glutamine (Life Technologies) at 37° C. in the presence of 5% CO 2 . The pEGFP-N2-ovPD-1 or pEGFP-N2 (negative control) was introduced into COS-7 cells at 0.4 μg/cm 2  using Lipofectamine 2000 (Invitrogen). The cells were cultured for 48 hours (ovPD-1-EGFP expressing cells). In order to confirm the expression of ovine PD-1 in the thus prepared expressing cells, intracellular localization of EGFP was visualized with an all-in-one fluorescence microscope BZ-9000 (KEYENCE). 
     1.3. Reactivity of Rat Anti-Bovine PD-1 Antibody 5D2 with Ovine PD-1 (FIG.  9 ) 
     It was confirmed by flow cytometry that rat anti-bovine PD-1 monoclonal antibody cross-reacts with ovine PD-1. Ovine PD-1 -EGFP expressing COS-7 cells were blocked with 10% inactivated goat serum (Invitrogen)-supplemented PBS at room temperature for 15 min and reacted with 10 μg/ml of rat anti-bovine PD-1 antibody 5D2 at room temperature for 30 min. After washing, the cells were reacted with APC-labeled anti-rat Ig goat antibody (Beckman Coulter) at room temperature for 30 min. As a negative control antibody, rat IgG2a (κ) isotype control (BD Bioscience) was used. For analysis, FACS Verse (BD Bioscience) was used. For every washing operation and dilution of antibodies, 1% bovine serum albumin (Sigma-Aldrich)-supplemented PBS was used. 
     The experimental results are shown in  FIG. 9 . It was confirmed that rat anti-bovine PD-1 antibody 5D2 binds to ovine PD-1 expressing cells. 
     1.4. Reactivity of Rat Anti-Bovine PD-1 Antibody 5D2 with Water Buffalo Lymphocytes (FIG.  10 ) 
     Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of water buffalo ( Bubalus ubalis ; Asian water buffalo) by density gradient centrifugation using Percoll (GE Healthcare). The isolated water buffalo PBMCs were suspended in RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), penicillin 200 U/ml, streptomycin 200 μg/ml and 0.01% L-glutamine (Life Technologies). Cell density was adjusted to 2×10 6  cells/ml. To these PBMCs, phorbol 12-myristate acetate (PMA) 20 ng/ml and ionomvcin 1 μg/ml (Sigma-Aldrich) were added, followed by a 2-day culture at 37° C. under 5% CO 2 . Cultured PBMCs were harvested and blocked with 10% inactivated goat serum (Invitrogen)-supplemented. PBS at room temperature for 15 min. Then, rat anti-bovine PD-1 antibody 5D2 and mouse anti-bovine CD8 antibody (38.65, AbD Serotec) were reacted at room temperature for 30 min. As a negative control, rat IgG2a (κ) isotype control (BD Bioscience) was used. After washing, APC-labeled goat anti-rat Ig antibody (Beckman Coulter) and PE-labeled goat anti mouse IgG antibody (Beckman Coulter) were reacted at room temperature for 30 min. After further washing. Alexa Flour488-labeled mouse anti-bovine CD4 antibody (CC30, AbD Serotec) and PE/Cy7-labeled anti-bovine IgM mouse antibody (IL-A30, AbD Serotec) were reacted at room temperature for 30 min. For antibody labeling, Zenon Mouse IgG1 Labeling Kits (Life Technologies) or Lightning-Link Kits (Innova Biosciences) was used. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibodies, 10% inactivated goat serum (Invitrogen)-supplemented PBS was used. 
     The experimental results are shown in  FIG. 10 . Rat anti-bovine PD-1 antibody 5D2 strongly bound to water buffalo CD4 +  T cells (IgM − CD4 + ) and CD8 +  T cells (IgM − CD8 + ) that had been activated by PMA/ionomycin stimulation. 
     Example 3 
     Binding to Bovine Fcγ Receptors of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody Having Wild-Type or Mutated Bovine IgG1 
     Introduction 
     The present inventors have established a rat-bovine chimeric anti-bovine PD-1 antibody in Example 1 with a view to establishing a novel therapy for bovine infections. In the process, mutations were added to putative binding sites for Fcγ receptors in bovine IgG1 CH2 domain in order to suppress ADCC activity mediated by the chimeric antibody ( FIGS. 1 and 11 ). In the subject Example, in order to examine the effect of these amino acid mutations, the present inventors prepared rat-bovine chimeric anti-bovine PD-1 antibodies having mutated bovine IgG1 (“IgG1 ADCC-” described above) and wild-type bovine IgG1 (“IgG1 WT”), respectively, and confirmed their binding to known bovine Fcγ receptors. 
     Materials, Methods and Experimental Results 
     2.1. Preparation of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody Expressing Vector 
     Rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 having wild-type bovine IgG1 (IgG1 WT) or mutated bovine IgG1 (IgG1 ADCC- described above) was established. 
     An expression plasmid encoding rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 having mutated bovine IgG1 (IgG1 ADCC-) was prepared according to the procedures described in Example 1 (SEQ ID NOS: 9 and 10 (amino acid sequences), SEQ II) NOS: 14 and 15 (nucleotide sequences after codon optimization)). It should be noted that in order to suppress ADCC activity, the bovine IgG1 used in ch5D2 IgG1 ADCC- had mutations added to the putative binding sites for Fcγ receptors in CH2 domain (see  FIGS. 1 and 11  for amino acid numbers and mutations: 251 E→P, 252 L→V, 253 P→A, 254 G→deletion, 348 A→S, 349 P→S; Ikebuchi R, Konnai S, Okagawa I, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology, 142(4): 551-561; Aug. 2014). Hereinafter, the thus prepared plasmid is designated as pDN112-boPD-1 ch5D2 IgG1 ADCC-. 
     An expression plasmid encoding rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 having wild-type IgG1 (IgG1 WT) was prepared according to the procedures described below. First, in order to amplify the gene encoding the constant region of wild-type bovine IgG1. (GenBank accession number X62916), PCR was performed using a synthesized bovine PBMC-derived cDNA as a template and designed primers that have NheI and XbaI recognition sites added on the 5′ side (boIgG1 CH1 F and boIgG1 CH3 R). The amplified gene strand was digested with NheI (Takara) and XbaI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics), and cloned into pDN112-boPD-1ch5D2 IgG1 ADCC- that had been treated with the restriction enzymes in the same manner. Further, the resultant plasmid was purified with QIAGEN Plasmid Midi kit (Qiagen) and digested with NotI (Takara) and XbaI (Takara) to thereby obtain an expression cassette for ch5D2&#39;s light chain (SEQ ID NO: 9 (amino acid sequence), SEQ ID NO: 14 (nucleotide sequence)) and heavy chain (IgG1 WT) (SEQ ID NO: 75 (amino acid sequence), SEQ ID NO: 76 (nucleotide sequence)). This gene fragment was purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into the cloning site (NotI and XbaI restriction enzyme recognition sequences downstream of PCMV and between INRBG and PABGH) of expression vector pDC6 (kindly provided by Prof. S. Suzuki, Hokkaido University Research Center for Zoonosis Control) ( FIG. 12 ). The resultant expression plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at 30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pDC6-boPD-1ch5D2 IgG1 WT. 
     
       
         
           
               
               
            
               
                   
                 Primer (boIgG1 CH1 F): 
               
               
                   
                 (SEQ ID NO: 77) 
               
               
                   
                 CTAGCTAGCACCACAGCCCCGAAAGTCT 
               
               
                   
                   
               
               
                   
                 Primer (boIgG1 CH3 R): 
               
               
                   
                 (SEQ ID NO: 78) 
               
               
                   
                 TGCTCTAGATTATTTACCCGCAGACCTTAGA 
               
            
           
         
       
     
     2.2. Expression and Purification of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody 
     Thirty micrograms of pDC6-boPD-1ch5D2 IgG1 WT or pDN112-boPD-1ch5D2 IgG1 ADCC- was introduced into 7.5×10 7  Expi293F cells (Life Technologies) using Expifectamine (Life Technologies) and the transfected cells were then cultured under shaking for 5 to 7 days, followed by collection of a culture supernatant. Each chimeric antibody was purified from the culture supernatant using Ab Capcher Extra (ProteNova). An open column method was used for binding to resin; 1.5 M Glycine/3 M NaCl (pH 8.0) was used as equilibration buffer and wash buffer. As elution buffer, 0.1 M Glycine-HCl (pH 2.8) was used. As neutralization buffer, 1M Tris (pH 9.0) was used. The purified antibody was subjected to buffer exchange with PBS (pH 7.4) using PD-10 Desalting Column (GE Healthcare) and concentrated using Amicon Ultra-15 (50 kDa, Millipore). The thus purified chimeric antibody was passed through a 0.22 μm syringe filter (Pall Life Science) for sterilization and stored at 4° C. until use in experiments. The concentration of each chimeric antibody as purified was quantitatively determined with the absorbance (280 nm) measured with Nanodrop8000 Spectrophotometer (Thermo Fisher Scientific). 
     2.3. Confirmation of the Purity of Purified Rat-Bovine Chimeric Anti-Bovine PD-1 Antibodies (FIG.  13 ) 
     In order to confirm the purity of purified rat-bovine chimeric anti-bovine PD-1 antibodies (ch5D2 IgG1 WT and ch5D2 IgG1 ADCC-), antibody proteins were detected by SDS-PAGE and CBB staining. Each chimeric antibody purified was suspended in Laemmli Sample Buffer (Bio-Rad) and denatured at 9.5° C. for 5 min under reducing conditions (reduced with 2-mercaptoethaanol; Sigma-Aldrich) or under non-reducing conditions. The thus prepared samples were electrophoresed using SuperSep Ace 5%-20% gradient polyacrylamide gel (Wako). As molecular weight markers, Precision Plus Protein All Blue Standards (Bio-Rad) were used. After electrophoresis, the gel was stained with Quick-CBB kit (Wako) and decolored in distilled water. 
     The results are shown in  FIG. 13 . Bands of ch5D2IgG1 WT and ch5D2 IgG1 ADCC- were observed at predicted positions, that is, at 25 kDa (light chain) and 50 kDa (heavy chain) under reducing conditions and at 150 kDa under non-reducing conditions. 
     2.4. Construction of Soluble Bovine Fcγ Receptors (FcγRs) 
     Bovine FcγRI-His, FcγRIII-His and Fcγ2R-His expressing plasmids were constructed according to the procedures described below, in order to amplify the signal peptide and the extracellular region of bovine FcγRI, FcγRII, FcγRIII and Fcγ2R (GenBank accession numbers NM_174538, NM_174539, NM_001077402 and NM_001001138), primers were designed which had NotI and XhoI recognition sites added on the 5′ side (boFcγRI-His F and R; boFcγRIII-His F and R; or bocγ2R-His F and R) or NheI and EcoRV recognition sites added on the 5′ side (boFcγRIII-His F and R). A gene sequence encoding a 6×His tag was added to reverse primers. PCR was performed using a synthesized bovine PBMC-derived cDNA as a template. The respective PCR products were digested with NotI (Takara) and XhoI (Takara) (FcγRI-His, FcγRIII-His and Fcγ2R-His) or NheI (Takara) and EcoRV (Takara) (FcγRII-His), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCXN2.1(+) vector (Nivea H. Yamamura K, Miyazaki. J. Gene, 108(2): 193-199; Dec. 15, 1991; kindly provided by Dr. T. Yokomizo, Juntendo University Graduate School of Medicine). The resultant expression plasmids were purified with FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCXN2.1-boFcγRI-His, pCXN2.1-boFcγRII-His, pCXN2.1-boFcγRIII-His or pCXN2.1-boFcγ2R-His. 
     
       
         
           
               
            
               
                 Primer (boFcγRI-His F): 
               
               
                 (SEQ ID NO: 79) 
               
               
                 ATAAGAATGCGGCCGCCACCATGTGGCTCATAATAGCTCT 
               
               
                   
               
               
                 Primer (boFcγRI-His R): 
               
               
                 (SEQ ID NO: 80) 
               
               
                 GCCCTCGAGTTAATGGTGATGGTGATGGTGAGGAGTTGTTGACTGGAGGC 
               
               
                   
               
               
                 Primer (boFcγRII-His F): 
               
               
                 (SEQ ID NO: 81) 
               
               
                 ATAAGAATGCTAGCCACCATGGGGATCCCCTCATTCCT 
               
               
                   
               
               
                 Primer (boFcγRII-His R): 
               
               
                 (SEQ ID NO: 82) 
               
               
                 GCCGATATCTTAATGGTGATGGTGATGGTGCGATGAGGGGCCGCTCGAGC 
               
               
                   
               
               
                 Primer (boFcγRIII-His F): 
               
               
                 (SEQ ID NO: 83) 
               
               
                 ATAAGAATGCGGCCGCCACCATGTGGCAACTGCTACCACC 
               
               
                   
               
               
                 Primer (boFcγRIII-His R): 
               
               
                 (SEQ ID NO: 84) 
               
               
                 GCCCTCGAGTTAATGGTGATGGTGATGGTGGTGCCAAGGTAGAAAGAATG 
               
               
                   
               
               
                 Primer (boFcγ2R-His F): 
               
               
                 (SEQ ID NO: 85) 
               
               
                 ATAAGAATGCGGCCGCCACCATGGCCCCCACCCTCCCTGCCTTGCTCT 
               
               
                   
               
               
                 Primer (boFcγ2R-His R): 
               
               
                 (SEQ ID NO: 86) 
               
               
                 GCCCTCGAGTTAATGGTGATGGTGATGGTGATTCTGCATCGTGTAGTCTG 
               
            
           
         
       
     
     Soluble bovine FcγRI-His, FcγRII-His, FcγRIII-His and Fcγ2R-His expressing cells were prepared according to the procedures described below Briefly, 30 μg of pCXN2.1-boFcγRI-His, pCXN2.1-boFcγRII-His, pCXN2.1-boFcγRIII-His or pCXN2.1-boFcγ2R-His was introduced into 7.5×10 7  Expi293F cells (Life Technologies) using Expifectamine (Life Technologies) and the transfected cells were then cultured under shaking for 5 to 7 days, followed by collection of a culture supernatant. Recombinant proteins were purified from the culture supernatant using TALON Metal Affinity Resin (Clontech). After purification, the buffer was exchanged with PBS (pH 7.4) using Amicon Ultra-15 Centrifugal Filter Unit (10 kDa, Millipore), and the recombinant proteins were stored at −30° C. until use in experiments (bovine PD-1-His). The concentrations of purified bovine FcγRI-His, FcγRII-His, FcγRIII-His and Fcγ2R-His were quantitatively determined in terms of the absorbance (280 nm) measured with Nanodrop8000 Spectrophotometer (Thermo Fisher Scientific). 
     2.5. Binding to Bovine FcγRs of Rat-Bovine Chimeric Anti-Bovine PD-1 Antibody ch5D2 IgG1 WT and IgG1 ADCC- (FIG.  14 ) 
     Rat-bovine chimeric anti-bovine PD-1 antibody ch5D2 IgG1 WT or IgG1 ADCC- was immobilized on Nunc MaxiSorp ELISA plates (Nunc) at a final concentration of 50, 25, 12.5, 6.25, 3.12 or 1.5610 nM at 37° C. for 2 hr. Subsequently, each well was washed with 200  i d of 0.05% Tween 20-supplemented PBS (PBS-T) five times, followed by blocking with SuperBlock (PBS) Blocking Buffer (Thermo Fisher Scientific) at 37° C. for 30 min, Each well was washed again in the same manner. Then, bovine FcγRI-His, FcγRII-His, FcγRIII-His or Fcγ2R-His was added to each well at a final concentration of 10 μg/ml and reacted at 37° C. for 1 hr. After washing, anti-polyhistidine tag mouse monoclonal antibody (Abeam) was reacted at 37° C. for 30 min. Subsequently, each well was washed, and horseradish peroxidase-labeled anti-mouse IgG goat polyclonal antibody (MP Biomedicals) was reacted at 37° C. for 30 min. Each well was washed again, and then TMB One Component Substrate (Bethyl) was added for coloring. Thereafter, the enzyme reaction was terminated with 0.18 M dilute sulfuric acid, and absorbance (450 nm) was measured with Microplate Reader MTP-900 (Corona Electric), For every plate washing operation, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. 
     The experimental results are shown in  FIG. 14 . IgG1 WT strongly bound to bovine FcγRI-His and weakly bound to bovine FcγRII-His. On the other hand, IgG1 ADCC- did not bind to bovine FcγRI-His or FcγRII-His. Neither IgG1 WT nor IgG1 ADCC- bound to bovine FcγRIII-His or Fcγ2R-His. 
     All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety. 
     INDUSTRIAL APPLICABILITY 
     The anti-PD-1 antibody of the present invention is applicable to prevention and/or treatment of cancers and infections of animals. 
     
       
         
           
               
             
               
                   
               
               
                 SEQUENCE LISTING FREE TEXT 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 &lt;SEQ ID NO: 1&gt; 
               
               
                 SEQ ID NO: 1 shows the amino acid sequence of the light chain variable region (VL) of a rat 
               
               
                 anti-bovine PD-1 antibody, Underlined parts: CDR1, CDR2 and CDR3 in this order from the 
               
               
                 NH2 terminus. 
               
               
                 MKVPGRLLVLLFWIPASRSDVVLTQTPVSLSVFLGDQASISCRSS QSLEYSDGYTY LE 
               
               
                 WYLQKPGQSPQLLIYGVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYC FQAT   
               
               
                   HDPDT FGAGTKLELK 
               
               
                   
               
               
                 &lt;SEQ ID NO: 2&gt; 
               
               
                 SEQ ID NO: 2 shows the amino acid sequence of the heavy chain variable region (VH) of a 
               
               
                 rat anti-bovine PD-1 antibody, Underlined parts: CDR1, CDR2 and CDR3 in this order from 
               
               
                 the NH2 terminus. 
               
               
                 MAILVLLLCLVTIPHSVLSQVQLKETGPGLVQPTQTLSITCTVS GFSLTSYY IQWVRQT 
               
               
                 PGKGLEWMGF IRSGGST EYNSEFKSRLSINRDTSKNQVFLKMNSLKTEDTGVYYC AR   
               
               
                   TSSGYEGGFDY WGQGVMVTVSS 
               
               
                   
               
               
                 &lt;SEQ ID NO: 3&gt; 
               
               
                 SEQ ID NO: 3 shows the amino acid sequence of the light chain constant region (CL) of a 
               
               
                 bovine antibody (bovine Ig lambda, GenBank: X62917), 
               
               
                 QPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASK 
               
               
                 QSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS 
               
               
                   
               
               
                 &lt;SEQ ID NO: 4&gt; 
               
               
                 SEQ ID NO: 4 shows the amino acid sequence of the heavy chain constant region (CH) of a 
               
               
                 bovine antibody (bovine IgG1, modified from Gen:Bank: X62916). Mutated parts are 
               
               
                 underlined. Amino acid numbers and mutations: 123 E→P, 124 L→V, 125 P→A, 126 G→ 
               
               
                 deletion, 218 A→S, 219 P→S 
               
               
                 ASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVL 
               
               
                 QSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVDPTCKPSPCDCCPPP PVA   
               
               
                 GPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREE 
               
               
                 QFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLP SS IVRTISRTKGPAREPQVYVLA 
               
               
                 PPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADSSYFLYS 
               
               
                 KLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGK 
               
               
                   
               
               
                 &lt;SEQ ID NO: 5&gt; 
               
               
                 SEQ ID NO: 5 shows the nucleotide sequence of the VL of a rat anti-bovine PD-1 antibody. 
               
               
                 ATGAAAGTGCCTGGTAGGCTGCTGGTGCTGTTGTTTTGGATTCCAGCTTCCAGGAG 
               
               
                 TGATGTTGTGTTGACACAAACTCCAGTTTCCCTGTCTGTCACACTTGGAGATCAAG 
               
               
                 CTTCTATATCTTGCAGGTCTAGTCAGAGCCTGGAATATAGTGATGGATACACTTATT 
               
               
                 TGGAATGGTACCTACAGAAGCCAGGCCAGTCTCCACAGCTCCTCATCTATGGAGTT 
               
               
                 TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCATTGGCAGTGGGTCAGGGACAG 
               
               
                 ATTTCACCCTCAAGATCAGCAGAGTAGAGCCTGAGGACTTGGGAGTTTATTACTGC 
               
               
                 TTCCAAGCTACACATGATCCGGACACGTTTGGAGCTGGGACCAAGCTGGAACTGA 
               
               
                 AA 
               
               
                   
               
               
                 The nucleotide sequence of SEQ ID NO: 5 after codon optimization is shown 
               
               
                 in &lt;SEQ ID NO: 11&gt;. 
               
               
                 ATGAAGGTCCCTGGTAGGCTGCTGGTTCTCTTGTTCTGGATCCCTGCTTCCAGAAG 
               
               
                 TGACGTGGTGCTGACTCAAACACCAGTGAGTCTCAGTGTGACCCTCGGCGACCA 
               
               
                 GGCCTCCATTTCTTGCCGTAGCAGCCAGTCCTTGGAATACTCTGATGGTTATACTTA 
               
               
                 TCTGGAGTGGTACCTCCAGAAGCCCGGGCAGTCACCCCAGCTTCTTATCTACGGT 
               
               
                 GTGAGCAACAGATTTTCTGGGGTTCCTGATCGGTTTATTGGATCTGGATCCGGTAC 
               
               
                 CGACTTCACATTGAAAATTTCACGCGTCGAACCCGAGGATTTGGGGGTCTACTATT 
               
               
                 GCTTCCAAGCCACCCACGATCCCGACACCTTCGGCGCTGGCACTAAGCTGGAGCT 
               
               
                 GAAA 
               
               
                   
               
               
                 &lt;SEQ ID NO: 6&gt; 
               
               
                 SEQ ID NO: 6 shows the nucleotide sequence of the VH of a rat anti-bovine PD-1 antibody. 
               
               
                 ATGGCTATCCTGGTGCTGCTTCTCTGCCTGGTGACCATTCCACACTCTGTCTTGTCC 
               
               
                 CAGGTGCAGCTGAAGGAGACAGGACCTGGCCTGGTGCAACCAACACAGACCCTG 
               
               
                 TCCATCACATGTACTGTTTCTGGGTTCTCATTAACCAGCTATTATATACAGTGGGTTC 
               
               
                 GCCAGACTCCAGGAAAGGGACTAGAATGGATGGGATTTATACGGAGTGGTGGAAG 
               
               
                 CACAGAGTATAATTCAGAGTTCAAATCCCGACTTAGCATCAACAGGGACACCTCC 
               
               
                 AAGAACCAAGTTTTCTTAAAAATGAACAGTCTGAAAACAGAGGACACAGGCGTG 
               
               
                 TACTACTGTGCCAGAACCTCTTCGGGGTACGAAGGGGGTTTTGATTACTGGGGCC 
               
               
                 AAGGAGTCATGGTCACAGTCTCCTCA 
               
               
                   
               
               
                 The nucleotide sequence of SEQ ID NO: 6 after codon optimization is shown in &lt;SEQ ID 
               
               
                 NO: 12&gt;. 
               
               
                 ATGGCAATCCTCGTGTTGCTTCTGTGCTTGGTGACCATTCCACACTCTGTGCTTTCC 
               
               
                 CAGGTGCAGCTCAAGGAAACAGGGCCAGGACTCGTCCAACCTACACAAACCCTG 
               
               
                 TCAATCACCTGTACCGTATCCGGTTTTAGCCTCACCAGCTATTATATACAATGGGTG 
               
               
                 AGGCAGACCCCCGGGAAAGGACTGGAATGGATGGGCTTCATTCGCAGCGGTGGG 
               
               
                 AGTACCGAGTACAATAGCGAGTTTAAAAGTCGCTTGAGTATCAATAGAGATACTTC 
               
               
                 CAAGAATCAGGTGTTCTTGAAGATGAACTCCCTCAAGACCGAAGATACAGGGGTC 
               
               
                 TATTACTGCGCCAGGACCTCCAGTGGATATGAAGGAGGCTTTGATTATTGGGGGCA 
               
               
                 GGGCGTCATGGTAACTGTGAGCTCA 
               
               
                   
               
               
                 &lt;SEQ ID NO: 7&gt; 
               
               
                 SEQ ID NO: 7 shows the nucleotide sequence of the CL of a bovine antibody (bovine Ig 
               
               
                 lambda, GenBank: X62917). 
               
               
                 CAGCCCAAGTCCCCACCCTCGGTCACCCTGTTCCCGCCCTCCACGGAGGAGCTCA 
               
               
                 ACGGCAACAAGGCCACCCTGGTGTGTCTCATCAGCGACTTCTACCCGGGTAGCGT 
               
               
                 GACCGTGGTCTGGAAGGCAGACGGCAGCACCATCACCCGCAACGTGGAGACCAC 
               
               
                 CCGGGCCTCCAAACAGAGCAACAGCAAGTACGCGGCCAGCAGCTACCTGAGCCT 
               
               
                 GACGAGCAGCGACTGGAAATCGAAAGGCAGTTACAGCTGCGAGGTCACGCACGA 
               
               
                 GGGGAGCACCGTGACGAAGACAGTGAAGCCCTCAGAGTGTTCTTAG 
               
               
                   
               
               
                 The nucleotide sequence of SEQ ID NO: 7 after codon optimization is shown in &lt;SEQ ID 
               
               
                 NO: 13&gt;. 
               
               
                 CAACCCAAGTCACCACCATCCGTGACTCTGTTTCCTCCATCTACAGAGGAGCTGA 
               
               
                 ACGGAAACAAAGCTACCTTGGTGTGTCTCATCTCTGACTTTTACCCCGGATCTGTC 
               
               
                 ACTGTGGTATGGAAAGCAGATGGCAGCACAATAACCAGGAATGTTGAAACCACAC 
               
               
                 GAGCCTCCAAGCAGTCCAATAGTAAGTATGCCGCATCTTCATATCTGTCCCTTACAA 
               
               
                 GCTCAGACTGGAAATCCAAAGGCAGCTACAGTTGCGAGGTCACACATGAAGGCA 
               
               
                 GCACCGTGACAAAGACCGTAAAGCCATCTGAGTGTAGCTAG 
               
               
                   
               
               
                 &lt;SEQ ID NO: 8&gt; 
               
               
                 SEQ ID NO: 8 shows the nucleotide sequence (after codon optimization) of the CH of a 
               
               
                 bovine antibody (bovine IgG1, modified from GenBank: X62916). 
               
               
                 GCTAGCACCACAGCACCTAAAGTTTACCCTCTGTCTTCCTGCTGCGGCGACAAGT 
               
               
                 CTTCATCAACTGTTACTCTTGGATGCCTGGTCTCAAGTTACATGCCCGAGCCCGTG 
               
               
                 ACAGTGACCTGGAACTCAGGCGCTCTGAAGTCTGGAGTGCACACATTTCCAGCTG 
               
               
                 TGCTTCAGTCTAGCGGCCTGTATTCCCTCAGCTCTATGGTTACTGTACCTGGTAGCA 
               
               
                 CCAGCGGACAGACTTTCACCTGTAATGTTGCCCATCCCGCATCTTCTACCAAGGTC 
               
               
                 GATAAAGCCGTTGACCCCACTTGCAAACCATCCCCTTGTGATTGTTGTCCACCCCC 
               
               
                 TCCAGTGGCTGGCCCTTCCGTCTTCATTTTCCCTCCTAAACCTAAGGATACTCTGAC 
               
               
                 CATCTCAGGGACACCCGAGGTCACCTGTGTCGTCGTGGACGTGGGACATGACGAC 
               
               
                 CCAGAAGTCAAGTTCTCATGGTTCGTGGACGATGTGGAGGTGAACACAGCAACA 
               
               
                 ACAAAGCCCAGAGAAGAACAGTTTAACAGCACATATCGGGTGGTCAGCGCCTTGC 
               
               
                 GTATTCAGCACCAGGACTGGACTGGTGGCAAGGAGTTTAAGTGCAAGGTGCATAA 
               
               
                 CGAAGGTCTGCCCTCTTCTATAGTGAGAACTATCTCCCGAACTAAGGGCCCCGCTC 
               
               
                 GGGAGCCCCAGGTTTACGTCCTTGCTCCCCCTCAGGAGGAACTGAGTAAATCAAC 
               
               
                 CGTGAGTCTCACCTGTATGGTTACCTCATTTTACCCAGACTACATCGCCGTAGAGT 
               
               
                 GGCAGAGGAATGGACAGCCAGAGTCTGAGGACAAATACGGCACTACTCCTCCCC 
               
               
                 AACTGGATGCCGACTCTTCCTACTTCCTCTACTCCAAATTGCGAGTTGACCGGAAC 
               
               
                 TCATGGCAGGAGGGGGACACATACACATGCGTCGTTATGCACGAGGCCCTGCACA 
               
               
                 ACCATTACACCCAGAAGTCCACATCTAAAAGTGCAGGTAAGTAA 
               
               
                   
               
               
                 &lt;SEQ ID NO: 9&gt; 
               
               
                 SEQ ID NO: 9 shows the amino acid sequence of a chimeric light chain consisting of 
               
               
                 the VL of a rat anti-bovine PD-1 antibody and the CL of a bovine antibody. 
               
               
                 MKVPGRLLVLLFWIPASRSDVVLTQTPVSLSVFLGDQASISCRSSQSLEYSDGYTYLE 
               
               
                 WYLQKPGQSPQLLIYGVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYCFQAT 
               
               
                 HDPDTFGAGTKLELKQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKA 
               
               
                 DGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVFKFVKP 
               
               
                 SECS 
               
               
                   
               
               
                 &lt;SEQ ID NO: 10&gt; 
               
               
                 SEQ ID NO: 10 shows the amino acid sequence of a chimeric heavy chain consisting of the 
               
               
                 VH of a rat anti-bovine PD-1 antibody and the CH of a bovine antibody (bovine IgG1, 
               
               
                 modified from GenBank: X62916), 
               
               
                 MAILVLLLCLVTIPHSVLSQVQLKETGPGLVQPTQTLSITCTVSGFSLTSYYIQWVRQT 
               
               
                 PGKGLEWMGFIRSGGSTEYNSEFKSRLSINRDTSKNQVFLKMNSLKTEDTGVYYCAR 
               
               
                 TSSGYEGGFDYWGQGVMVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPE 
               
               
                 PVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKV 
               
               
                 DKAVDPTCKPSPCDCCPPPPVAGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEV 
               
               
                 KFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLP 
               
               
                 SSIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPE 
               
               
                 SEDKYGITPPQLDADSSYFLYSKLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTS 
               
               
                 KSAGK 
               
               
                   
               
               
                 &lt;SEQ ID NO: 14&gt; 
               
               
                 SEQ ID NO: 14 shows the nucleotide sequence (after codon optimization) of a chimeric light 
               
               
                 chain consisting of the VL of a rat anti-bovine PD-1 antibody and the CL of a bovine 
               
               
                 antibody. 
               
               
                 ATGAAGGTCCCTGGTAGGCTGCTGGTTCTCTTGTTCTGGATCCCTGCTTCCAGAAG 
               
               
                 TGACGTGGTGCTGACTCAAACACCAGTGAGTCTCAGTGTGACCCTCGGCGACCA 
               
               
                 GGCCTCCATTTCTTGCCGTAGCAGCCAGTCCTTGGAATACTCTGATGGTTATACTTA 
               
               
                 TCTGGAGTGGTACCTCCAGAAGCCCGGGCAGTCACCCCAGCTTCTTATCTACGGT 
               
               
                 GTGAGCAACAGATTTTCTGGGGTTCCTGATCGGTTTATTGGATCTGGATCCGGTAC 
               
               
                 CGACTTCACATTGAAAATTTCACGCGTCGAACCCGAGGATCTGGGGGTCTACTATT 
               
               
                 GCTTCCAAGCCACCCACGATCCCGACACCTTCGGCGCTGGCACTAAGCTGGAGCT 
               
               
                 GAAACAACCCAAGTCACCACCATCCGTGACTCTGTTTCCTCCATCTACAGAGGAG 
               
               
                 CTGAACGGAAACAAAGCTACCTTGGTGTGTCTCATCTCTGACTTTTACCCCGGATC 
               
               
                 TGTCACTGTGGTATGGAAAGCAGATGGCAGCACAATAACCAGGAATGTTGAAACC 
               
               
                 ACACGAGCCTCCAAGCAGTCCAATAGTAAGTATGCCGCATCTTCATATCTGTCCCTT 
               
               
                 ACAAGCTCAGACTGGAAATCCAAAGGCAGCTACAGTTGCGAGGTCACACATGAA 
               
               
                 GGCAGCACCGTGACAAAGACCGTAAAGCCATCTGAGTGTAGCTAG 
               
               
                   
               
               
                 &lt;SEQ ID NO: 15&gt; 
               
               
                 SEQ ID NO: 15 shows the nucleotide sequence (after codon optimization) of a chimeric 
               
               
                 heavy chain consisting of the VH of a rat anti-bovine PD-1 antibody and the CH of a bovine 
               
               
                 antibody (bovine IgG1, modified from GenBank: X62916). 
               
               
                 ATGGCAATCCTCGTGTTGCTTCTGTGCTTGGTGACCATTCCACACTCTGTGCTTTCC 
               
               
                 CAGGTGCAGCTCAAGGAAACAGGGCCAGGACTCGTCCAACCTACACAAACCCTG 
               
               
                 TCAATCACCTGTACCGTATCCGGTTTTAGCCTCACCAGCTATTATATACAATGGGTG 
               
               
                 AGGCAGACCCCCGGGAAAGGACTGGAATGGATGGGCTTCATTCGCAGCGGTGGG 
               
               
                 AGTACCGAGTACAATAGCGAGTTTAAAAGTCGCTTGAGTATCAATAGAGATACTTC 
               
               
                 CAAGAATCAGGTGTTCTTGAAGATGAACTCCCTCAAGACCGAAGATACAGGGGTC 
               
               
                 TATTACTGCGCCAGGACCTCCAGTGGATATGAAGGAGGCTTTGATTATTGGGGGCA 
               
               
                 GGGCGTCATGGTAACTGTGAGCTCAGCTAGCACCACAGCACCTAAAGTTTACCCT 
               
               
                 CTGTCTTCCTGCTGCGGCGACAAGTCTTCATCAACTGTTACTCTTGGATGCCTGGT 
               
               
                 CTCAAGTTACATGCCCGAGCCCGTGACAGTGACCTGGAACTCAGGCGCTCTGAAG 
               
               
                 TCTGGAGTGCACACATTTCCAGCTGTGCTTCAGTCTAGCGGCCTGTATTCCCTCAG 
               
               
                 CTCTATGGTTACTGTACCTGGTAGCACCAGCGGACAGACTTTCACCTGTAATGTrG 
               
               
                 CCCATCCCGCATCTTCTACCAAGGTCGATAAAGCCGTTGACCCCACTTGCAAACCA 
               
               
                 TCCCCTTGTGATTGTTGTCCACCCCCTCCAGTGGCTGGCCCTTCCGTCTTCATTTTC 
               
               
                 CCTCCTAAACCTAAGGATACTCTGACCATCTCAGGGACACCCGAGGTCACCTGTGT 
               
               
                 CGTCGTGGACGTGGGACATGACGACCCAGAAGTCAAGTTCTCATGGTTCGTGGAC 
               
               
                 GATGTGGAGGTGAACACAGCAACAACAAAGCCCAGAGAAGAACAGTTTAACAGC 
               
               
                 ACATATCGGGTGGTCAGCGCCTTGCGTATTCAGCACCAGGACTGGACTGGTGGCA 
               
               
                 AGGAGTTTAAGTGCAAGGTGCATAACGAAGGTCTGCCCTCTTCTATAGTGAGAACT 
               
               
                 ATCTCCCGAACTAAGGGCCCCGCTCGGGAGCCCCAGGTTTACGTCCTTGCTCCCC 
               
               
                 CTCAGGAGGAACTGAGTAAATCAACCGTGAGTCTCACCTGTATGGTTACCTCATTT 
               
               
                 TACCCAGACTACATCGCCGTAGAGTGGCAGAGGAATGGACAGCCAGAGTCTGAG 
               
               
                 GACAAATACGGCACTACTCCTCCCCAACTGGATGCCGACTCTTCCTACTTCCTCTA 
               
               
                 CTCCAAATTGCGAGTTGACCGGAACTCATGGCAGGAGGGGGACACATACACATGC 
               
               
                 GTCGTTATGCACGAGGCCCTGCACAACCATTACACCCAGAAGTCCACATCTAAAA 
               
               
                 GTGCAGGTAAGTAA 
               
               
                   
               
               
                 &lt;SEQ ID NO: 16&gt; 
               
               
                 SEQ ID NO: 16 shows the amino acid sequence ( QSLEYSDGYTY ) of CDR1 of the VL of 
               
               
                 rat anti-bovine PD-1 antibody 5D2. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 17&gt; 
               
               
                 SEQ ID NO: 17 shows the amino acid sequence ( FQATHDPDT ) of CDR3 of the VL of rat 
               
               
                 anti-bovine PD-1 antibody 5D2. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 18&gt; 
               
               
                 SEQ ID NO: 18 shows the amino acid sequence ( GFSLTSYY ) of CDR1 of the VH of rat 
               
               
                 anti-bovine PD-1 antibody 5D2. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 19&gt; 
               
               
                 SEQ ID NO: 19 shows the amino acid sequence ( IRSGGST ) of CDR2 of the VH of rat anti- 
               
               
                 bovine PD-1 antibody 5D2. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 20&gt; 
               
               
                 SEQ ID NO: 20 shows the amino acid sequence ( ARTSSGYEGGFDY ) of CDR3 of the VH 
               
               
                 of rat anti-bovine PD-1 antibody 5D2. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 21&gt; 
               
               
                 SEQ ID NO: 21 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG1 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 22&gt; 
               
               
                 SEQ ID NO: 22 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG1 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 23&gt; 
               
               
                 SEQ ID NO: 23 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG1 variant 3). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 24&gt; 
               
               
                 SEQ ID NO: 24 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG2 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 25&gt; 
               
               
                 SEQ ID NO: 25 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG2 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 26&gt; 
               
               
                 SEQ ID NO: 26 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG2 variant 3). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 27&gt; 
               
               
                 SEQ ID NO: 27 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG3 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 28&gt; 
               
               
                 SEQ ID NO: 28 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG3 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 29&gt; 
               
               
                 SEQ ID NO: 29 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG1 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 30&gt; 
               
               
                 SEQ ID NO: 30 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG1 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 31&gt; 
               
               
                 SEQ ID NO: 31 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG1 variant 3). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 32&gt; 
               
               
                 SEQ ID NO: 32 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG2 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 33&gt; 
               
               
                 SEQ ID NO: 33 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG2 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 34&gt; 
               
               
                 SEQ ID NO: 34 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG2 variant 3). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 35&gt; 
               
               
                 SEQ ID NO: 35 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG3 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 36&gt; 
               
               
                 SEQ ID NO: 36 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody 
               
               
                 (IgG3 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 37&gt; 
               
               
                 SEQ ID NO: 37 shows the amino acid sequence of the CH (CH1-CH3) of an ovine antibody 
               
               
                 (IgG1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 38&gt; 
               
               
                 SEQ ID NO: 38 shows the nucleotide sequence of the CH (CH1-CH3) of an ovine antibody 
               
               
                 (IgG1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 39&gt; 
               
               
                 SEQ ID NO: 39 shows the amino acid sequence of the CH (CH1-CH3) of an ovine antibody 
               
               
                 (IgG2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 40&gt; 
               
               
                 SEQ ID NO: 40 shows the nucleotide sequence of the CH (CH1-CH3) of an ovine antibody 
               
               
                 (IgG2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 41&gt; 
               
               
                 SEQ ID NO: 41 shows the amino acid sequence of the light chain (Ig kappa (CK)) constant 
               
               
                 region of an ovine antibody. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 42&gt; 
               
               
                 SEQ ID NO: 42 shows the nucleotide sequence of the light chain (Ig kappa (CK)) constant 
               
               
                 region of an ovine antibody. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 43&gt; 
               
               
                 SEQ ID NO: 43 shows the amino acid sequence of the light chain (Ig lambda (CL)) constant 
               
               
                 region of an ovine antibody. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 44&gt; 
               
               
                 SEQ ID NO: 44 shows the nucleotide sequence of the light chain (Ig lambda (CL)) constant 
               
               
                 region of an ovine antibody. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 45&gt; 
               
               
                 SEQ ID NO: 45 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo 
               
               
                 antibody (presumed to be IgG1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 46&gt; 
               
               
                 SEQ ID NO: 46 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo 
               
               
                 antibody (presumed to be IgG1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 47&gt; 
               
               
                 SEQ ID NO: 47 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo 
               
               
                 antibody (presumed to be IgG2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 48&gt; 
               
               
                 SEQ ID NO: 48 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo 
               
               
                 antibody (presumed to be IgG2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 49&gt; 
               
               
                 SEQ ID NO: 49 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo 
               
               
                 antibody (presumed to be IgG3). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 50&gt; 
               
               
                 SEQ ID NO: 50 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo 
               
               
                 antibody (presumed to be IgG3). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 51&gt; 
               
               
                 SEQ ID NO: 51 shows the amino acid sequence of the constant region of the light chain 
               
               
                 (presumed to be Ig lambda) of a water buffalo antibody. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 52&gt; 
               
               
                 SEQ ID NO: 52 shows the nucleotide sequence of the constant region of the light chain 
               
               
                 (presumed to be Ig lambda) of a water buffalo antibody, 
               
               
                   
               
               
                 &lt;SEQ ID NO: 53&gt; 
               
               
                 SEQ ID NO: 53 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody 
               
               
                 (IgG4 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 54&gt; 
               
               
                 SEQ ID NO: 54 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody 
               
               
                 (IgG4 variant 1). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 55&gt; 
               
               
                 SEQ ID NO: 55 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody 
               
               
                 (IgG4 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 56&gt; 
               
               
                 SEQ ID NO: 56 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody 
               
               
                 (IgG4 variant 2). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 57&gt; 
               
               
                 SEQ ID NO: 57 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody 
               
               
                 (IgG4 variant 3). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 58&gt; 
               
               
                 SEQ ID NO: 58 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody 
               
               
                 (IgG4 variant 31). 
               
               
                   
               
               
                 &lt;SEQ ID NO: 59&gt; 
               
               
                 SEQ ID NO: 59 shows the amino acid sequence of the CL of a human antibody. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 60&gt; 
               
               
                 SEQ ID NO: 60 shows the nucleotide sequence of the CL of a human antibody. 
               
               
                   
               
               
                 &lt;SEQ ID NOS: 61-74&gt; 
               
               
                 SEQ ID NOS: 61-74 show the nucleotide sequences of primers boPD-1-myc F, boPD-1-myc 
               
               
                 R, boPD-L1-EGFP F, boPD-L1-EGFP R, boPD-1-His F, boPD-1-His R, ovPD-1 CDS F, 
               
               
                 ovPD-1 CDS R, buPD-1 CDS F1, buPD-1 CDS R1, buPD-1 CDS F2, buPD-1 CDS R2, 
               
               
                 ovPD-1-EGFP F and ovPD-1-EGFP R in this order. 
               
               
                   
               
               
                 &lt;SEQ ID NO: 75&gt; 
               
               
                 SEQ ID NO: 75 shows the nucleotide sequence of a chimeric heavy chain consisting of the 
               
               
                 VH of a rat anti-bovine PD-1 antibody and the CH of a bovine antibody (bovine IgG1, 
               
               
                 GenBank: X62916). 
               
               
                 ATGGCAATCCTCGTGTTGCTTCTGTGCTTGGTGACCATTCCACACTCTGTGCTTTCC 
               
               
                 CAGGTGCAGCTCAAGGAAACAGGGCCAGGACTCGTCCAACCTACACAAACCCTG 
               
               
                 TCAATCACCTGTACCGTATCCGGTTTTAGCCTCACCAGCTATTATATACAATGGGTG 
               
               
                 AGGCAGACCCCCGGGAAAGGACTGGAATGGATGGGCTTCATTCGCAGCGGTGGG 
               
               
                 AGTACCGAGTAGAATAGCGAGTTTAAAAGTCGCTTGAGTATCAATAGAGATACTTC 
               
               
                 CAAGAATCAGGTGTTCTTGAAGATGAACTCCCTCAAGACCGAAGATACAGGGGTC 
               
               
                 TATTACTGCGCCAGGACCTCCAGTGGATATGAAGGAGGCTTTGATTATTGGGGGCA 
               
               
                 GGGCGTCATGGTAACTGTGAGCTCAGCCTCCACCACAGCCCCGAAAGTCTACCCT 
               
               
                 CTGAGTTCTTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGGGCTGCCTGG 
               
               
                 TCTCCAGCTACATGCCCGAGCCGGTGACCGTGACCTGGAACTCGGGTGCCCTGAA 
               
               
                 GAGCGGCGTGCACACCTTCCCGGCTGTCCTTCAGTCCTCCGGGCTGTACTCTCTCA 
               
               
                 GCAGCATGGTGACCGTGCCCGGCAGCACCTCAGGACAGACCTTCACCTGCAACGT 
               
               
                 AGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGGCTGTTGATCCCACATGCAA 
               
               
                 ACCATCACCCTGTGACTGTTGCCCACCCCCTGAGCTCCCCGGAGGACCCTCTGTC 
               
               
                 TTCATCTTCCCACCGAAACCCAAGGACACCCTCACAATCTCGGGAACGCCCGAGG 
               
               
                 TCACGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAGGTGAAGTTCTCCTG 
               
               
                 GTTCGTGGACGACGTGGAGGTAAACACAGCCACGACGAAGCCGAGAGAGGAGC 
               
               
                 AGTTCAACAGCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCACCAGGACTG 
               
               
                 GACTGGAGGAAAGGAGTTCAAGTGCAAGGTCCACAACGAAGGCCTCCCGGCCCC 
               
               
                 CATCGTGAGGACCATCTCCAGGACCAAAGGGCCGGCCCGGGAGCCGCAGGTGTAT 
               
               
                 GTCCTGGCCCCACCCCAGGAAGAGCTGAGCAAAAGCACGGTCAGCCTCACCTGC 
               
               
                 ATGGTCACCAGCTTCTACCCAGACTACATCGCCGTGGAGTGGCAGAGAAACGGGC 
               
               
                 AGCCTGAGTCGGAGGACAAGTACGGCACGACCCCGCCCCAGCTGGACGCCGACA 
               
               
                 GCTCCTACTTCCTGTACAGCAAGCTCAGGGTGGACAGGAACAGCTGGCAGGAAG 
               
               
                 GAGACACCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAATCACTACACGCA 
               
               
                 GAAGTCCACCTCTAAGTCTGCGGGTAAATAA 
               
               
                   
               
               
                 &lt;SEQ ID NO: 76&gt; 
               
               
                 SEQ ID NO: 76 shows the amino acid sequence of a chimeric heavy chain consisting of the 
               
               
                 VH of a rat anti-bovine PD-1 antibody and the CH of a bovine antibody (bovine IgG1, 
               
               
                 GenBank: X62916). 
               
               
                 MAILVLLLCLVTIPHSVLSQVQLKETGPGLVQPTQTLSITCTVSGFSLTSYYIQWVRQT 
               
               
                 PGKGLEWMGFIRSGGSTEYNSEFKSRLSINRDTSKNQVFLKMNSLKTEDTGVYYCAR 
               
               
                 TSSGYEGGFDYWGQGVMVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPE 
               
               
                 PVTVTWNSGALKSGYHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKV 
               
               
                 DKAVDPTCKPSPCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPE 
               
               
                 VKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGL 
               
               
                 PAPIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQP 
               
               
                 ESEDKYGTTPPQLDADSSYFLYSKLRVDRNSWQEGDTYTCVVMHEALHNHYTQKST 
               
               
                 SKSAGK 
               
               
                   
               
               
                 &lt;SEQ ID NOS: 77-86&gt; 
               
               
                 SEQ ID NOS: 77-86 show the nucleotide sequences of primers boIgG1 CH1 F, boIgG1 CH3 
               
               
                 R, boFcγRI-His F, boFcγRI-His R, boFcγRII-His F, boFcγRII-His R, boFcγRIII-His F, 
               
               
                 boFcγRIII-His R, boFcγ2R-His F and boFcγ2R-His R in this order.