Patent Publication Number: US-2004053831-A1

Title: Veterinary immunisation vectors

Description:
[0001] This invention relates to novel C3d polypeptides, or fragments thereof, and to nucleic acid sequences encoding such polypeptides or fragments. The invention also relates to genetic constructs comprising genetic sequences encoding species-specific polypeptides designed to enhance the immunogenicity of antigens in non-human species and to methods for the generation of such constructs.  
       [0002] The invention further relates to the use of variant nucleic acid sequences to encode species-specific C3d polypeptides which, when used to express tandem arrays of the polypeptide show enhanced stability, leading to high level expression in eukaryotic and prokaryotic cell expression systems. When incorporated into a DNA immunization vector or a recombinant live organism vaccine, such sequences have reduced risk of undergoing homologous recombination with genomic DNA compared to wild-type sequences, thus reducing the risk of potentially damaging integration events.  
       [0003] A species-specific polypeptide linked to an antigen, or nucleic acid encoding the same may be administered as part of a prophylactic or therapeutic vaccine formulation to any non-human vertebrate (the host species), or administered with the intention of raising specific antibodies to the antigen in the host species. Such antigens may be derived from any organism including the host species. The species-specific polypeptides comprise or consist of tandem arrays of a polypeptide which occurs naturally in the host species and which has immunostimulatory properties. Examples of such polypeptides include polypeptides derived from the complement system, as described below. Such tandem arrays, when linked to an antigen may enhance humoral responses to the antigen by several orders of magnitude.  
       [0004] A number of naturally occurring immune modulators, such as cytokines, have been proposed for inclusion into DNA immunization vectors to be expressed concurrently with the antigen (reviewed by Leitner et al., 1999 Vaccine 18: 765-77). The use of naked DNA as an immunogen has raised concerns about the potential for its integration into the genome of the host species and the possibility of insertional mutagenesis resulting in the inactivation of tumor suppressor genes or the activation of oncogenes (reviewed by Nicholls et al., 1995 Ann N Y Acad Sci 772: 30-9). Such concerns apply equally to recombinant live organisms used as vaccines, many of which undergo rounds of self-replication in the host species. Although the studies reviewed by Nicholls et al., (1995) have shown integration to be a low frequency occurrence with plasmids containing sequences unrelated to the host species the inclusion of genes derived from the genome of the host species increases this risk significantly.  
       [0005] The complement system consists of a set of serum proteins that are important in the response of the immune system to foreign antigens. The complement system becomes activated when its primary components are cleaved and the products, alone or with other proteins, activate additional complement proteins resulting in a proteolytic cascade. Activation of the complement system leads to a variety of responses including increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, opsonisation of foreign particles, direct killing of cells and tissue damage. Activation of the complement system may be triggered by antigen-antibody complexes (the classical pathway) or a normal slow activation may be amplified in the presence of cell walls of invading organisms such as bacteria and viruses (the alternative pathway).  
       [0006] The complement system interacts with the cellular immune system through a specific pathway involving C3, a protein central to both classical and alternative pathways. The proteolytic activation of C3 gives rise to a large fragment (C3b) and exposes a chemically reactive internal thiolester linkage which can react covalently with external nucleophiles such as the cell surface proteins of invading organisms or foreign cells. As a result, the potential antigen is ‘tagged’ with C3b and remains attached to that protein as it undergoes further proteolysis to iC3b and C3d,g. The latter fragments are, respectively, ligands for the complement receptors CR3 and CR2. Thus the labelling of antigen by C3b can result in a targeting mechanism for cells of the immune system bearing these receptors.  
       [0007] That such targeting is important for augmentation of the immune response is first shown by experiments in which mice were depleted of circulating C3 and then challenged with an antigen (sheep erythrocytes). Removal of C3 reduced the antibody response to this antigen. (M. B. Pepys, J.Exp.Med, 140, 126-145, 1974). The role of C3 was confirmed by studies in animals genetically deficient in either C3 or the upstream components of the complement cascade which generate C3b, i.e. C2 and C4, (J. M. Ahearn &amp; D. T. Fearon, Adv.Immunol. 46, 183-219, 1989). More recently, it has been shown that linear conjugation of a model antigen with more than two copies of the murine C3d fragment sequence resulted in a very large (1000-10000-fold) increase in antibody response in mice compared with unmodified antigen controls (P. W. Dempsey et al, Science, 271: 348-350, 1996; WO96/17625, PCT/GB95/02851). The increase could be produced without the use of conventional adjuvants such as Freund&#39;s complete adjuvant. The mechanism of this remarkable effect was demonstrated to be high-affinity binding of the multivalent C3d construct to CR2 on B-cells, followed by co-ligation of CR2 with another B-cell membrane protein, CD19, and with membrane-bound immunoglobulin to generate a signal to the B-cell nucleus.  
       [0008] In the experiments of Dempsey et al, (1996) the unmodified antigen control and linear fusions with one or two C3d domains were prepared by transfection of the appropriate coding plasmids into L cells followed by the selection of high-expressing clones. The most immunogenic construct, that with three C3d units, had to be expressed transiently in COS cells and this procedure gave a very poor yield of the fusion protein. In part, the low yield could be attributed to the generation of species containing the antigen but with lower molecular weights, corresponding to fewer than three C3d units. It was unclear from the published work of Dempsey et al whether the latter molecules originated by proteolysis of the three-C3d construct or whether they were due to a recombination event in vivo.  
       [0009] Using another expression system but the same C3d constructs as Dempsey et al, we obtained evidence that the generation of molecules with &lt;3 C3d units from DNA encoding 3× C3d repeats is due to loss of one or more C3d units by homologous recombination and not due to post-translational processing (see WO99/35260) and described methods for the generation and selection of stable variant genes resistant to homologous recombination.  
       [0010] The present invention is defined in the appended independent claims. Preferred features of the invention are specified in the subclaims.  
       [0011] This invention may be used in any context where a nucleic acid sequence is included in a medicament where the sequence of the nucleic acid is homologous to a sequence in the genome of the recipient human or animal host. These may be used in the context of gene therapy, therapeutic or prophylactic vaccination of other therapeutic strategies in which nucleic acid forms part of the medicament. It is particularly useful for, but is not restricted to, DNA immunization vectors encoding proteins with immunopotentiating properties derived from the complement system.  
       [0012] Preferred embodiments of this invention related specifically to an immunostimulatory component of the complement system, and the use of species-specific components in veterinary vaccines for non-human vertebrate species or to raise antibodies in non-human vertebrate species.  
       [0013] The present invention provides:  
       [0014] i) a general method for cloning C3d from non-human vertebrates and the construction of variant DNA sequences encoding identical C3d units which can be ligated in tandem with each other with or without the native (wild-type) C3d DNA sequence and may be stably maintained in prokaryotic and eukaryotic expression vectors to produce oligomers of at least two or three copies of species-specific C3d at commercially viable levels; and  
       [0015] ii) the use of the oligomers in combination with antigens as vaccines or the nucleic acid sequence encoding the oligomers in DNA immunization vectors or in recombinant live organisms with reduced capacity for homologous integration into host genomic DNA.  
       [0016] The present invention also provides:  
       [0017] 1. Novel C3d cDNA sequences from non-human vertebrate species, and methods of cloning such sequences.  
       [0018] 2. Novel synthetic DNA sequences encoding oligomers of species-specific C3d where the polypeptide sequence of each unit of the C3d is identical, but the DNA encoding each unit is different, and methods of constructing such sequences.  
       [0019] 3. High-level expression of oligomers of species-specific C3d in prokaryotic and eukaryotic systems and maintenance of stable recombinant expression vector stocks.  
       [0020] 4. Use of variant C3d genes encoding species-specific C3d fused to antigen in a DNA immunization vector.  
       [0021] 5. Use of variant C3d genes encoding species-specific C3d fused to antigen in a recombinant live organism.  
       [0022] The present invention also provides a process for preparing an oligomeric polypeptide in vitro or in vivo comprising: constructing an expression vector, which may be a DNA vector or a recombinant live organism encoding the oligomeric polypeptide; introducing the expression vector into a recombinant host cell in vitro or a host organism in vivo; and culturing the recombinant host cell or host organism under conditions for expression of the polypeptide.  
       [0023] The process may further comprise amplifying species-specific nucleic acid encoding a C3d polypeptide from tissue derived from a vertebrate species. The process may further comprise recovering the polypeptide.  
       [0024] The present invention also provides a process for preparing an nucleic acid encoding a C3d polypeptide which comprises: amplifying species-specific nucleic acid encoding a C3d polypeptide from tissue derived from a vertebrate species; preparing a replicable expression vector from the amplified nucleic acid which encodes the C3d polypeptide; transforming a host cell with the vector; culturing the transformed host cell under conditions for replication of the expression vector; and recovering the expression vector in a form suitable for DNA immunization.  
       [0025] There is also provided a linear DNA concatameter encoding the oligomeric polypeptide.  
       [0026] Processes of the invention may be performed using conventional recombinant techniques such as described in Sambrook et al., Molecular Cloning: A laboratory manual 2nd Edition. Cold Spring Harbor Laboratory Press (1989) and DNA Cloning vols I, II and III (D. M. Glover ed., IRL Press Ltd).  
       [0027] The invention also provides a process for preparing the linear DNA concatamer by condensing appropriate mono-, di- or oligomeric nucleotide units.  
       [0028] The preparation may be carried out chemically, enzymatically, or by a combination of the two methods, in vitro or in vivo as appropriate. Thus, the linear DNA concatamer may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Roberts et al., in Biochemistry 1985, 24, 5090-5098.  
       [0029] The DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerisation, or by a combination of these methods.  
       [0030] Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70° C., generally in a volume of 50 μl or less with 0.1-10 μg DNA. Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase 1 (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37° C., generally in a volume of 50 μl or less. Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4° C. to 37° C., generally in a volume of 50 μl or less.  
       [0031] The chemical synthesis of the linear DNA concatamer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in ‘Chemical and Enzymatic Synthesis of Gene Fragments—A Laboratory Manual’ (ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M. J. Gait, H. W. D. Matthes M. Singh, B. S. Sproat and R. C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M. D. Matteucci and M. H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185;S. P. Adams et al., Journal of the American Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J. McMannus and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal, 1984, 3, 801. Preferably an automated DNA synthesiser (for example, Applied Biosystems 381A Synthesiser) is employed.  
       [0032] The linear DNA concatamer is preferably prepared by ligating two or more DNA molecules which together comprise a DNA sequence encoding the oligomeric polypeptide. The DNA molecules may be obtained by digestion with suitable restriction enzymes of vectors carrying the required coding sequences.  
       [0033] The precise structure of the DNA molecules and the way in which they are obtained depends upon the structure of the desired product. A linear DNA concatamer encoding the oligomeric polypeptide may be constructed using a variety of methods including chemical synthesis of DNA oligonucleotides, enzymatic polymerisation, restriction enzyme digestion and ligation.  
       [0034] Expression of the oligomeric polypeptide encoded by the linear DNA concatamer in a recombinant host cell or in vivo by a recipient of a DNA immunisation vector may be carried out by means of a replicable expression vector capable, in the host cell or in vivo, of expressing the polypeptide from the DNA polymer.  
       [0035] The replicable expression vector may be prepared by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment, encode the polypeptide, under ligating conditions.  
       [0036] Ligation of the linear segment and more than one DNA molecule may be carried out simultaneously or sequentially as desired. Thus, the linear DNA concatamer may be preformed or formed during the construction of the vector, as desired.  
       [0037] The choice of vector will be determined in part by the host cell, which may be prokaryotic, such as  E. coli , mammalian, such as mouse C127, mouse myeloma, Chinese hamster ovary, or other eukaryotic (fungi e.g. filamentous fungi or unicellular yeast or an insect cell such as Drosophila or Spodoptera). The host cell may also be in a transgenic animal or a human or animal recipient of a DNA immunization vector. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses derived from, for example, baculoviruses, vaccinia, adenovirus and herpesvirus.  
       [0038] The linear DNA concatamer may be assembled into vectors designed for isolation of stable transformed mammalian cell lines expressing the fragment e.g. bovine papillomavirus vectors in mouse C127 cells, or amplified vectors in Chinese hamster ovary cells (DNA Cloning Vol. II D. M. Glover ed. IRL Press 1985; Kaufman, R. J. et al.. Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G. N. and Hamer, D. H. Proceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D. V. et al. European Patent Application No. 0093619, 1983).  
       [0039] The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Sambrook et al., cited above. Polymerisation and ligation may be performed as described above for the preparation of the linear DNA concatamer. Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70° C., generally in a volume of 50 μl or less with 0.1-10 μg DNA.  
       [0040] A recombinant host cell may be prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Sambrook et al., cited above, or “DNA Cloning” Vol. II, D. M. Glover ed., IRL Press Ltd, 1985.  
       [0041] The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as  E. coli , may be treated with a solution of CaCl 2  (Cohen et al., Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl 2 , potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol or by electroporation as for example described by Bio-Rad Laboratories, Richmond, Calif., USA, manufacturers of an electroporator. Eukaryotic cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells or by using cationic liposomes.  
       [0042] DNA immunization vectors may be administered as naked DNA or contained within a viral particle by injection or by other means of delivery including aqueous or non-aqueous formulations via transdermal or mucosal routes.  
       [0043] The invention also provides a host cell transformed with a replicable expression vector of the invention.  
       [0044] Culturing the transformed host cell under conditions for expression of the linear DNA concatamer may be carried out conventionally, as described in, for example, Sambrook et al., and “DNA Cloning” cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45° C.  
       [0045] An oligomeric polypeptide of the invention may be recovered by conventional methods. Thus, where the host cell is bacterial such as  E. coli  and the oligomeric polypeptide is expressed intracellularly, it may be lysed physically, chemically or enzymatically and the oligomeric polypeptide isolated from the resulting lysate. Where the host cell is eukaryotic, the oligomeric polypeptide may be isolated from the nutrient medium. Where the host cell is in a transgenic animal the polypeptide may be recovered from the natural secretory pathways (e.g. where the polypeptide is secreted in the milk of a female transgenic animal). Where the host cell is in a human or animal recipient of a DNA immunization vector or gene therapy vector the oligomeric polypeptide would not normally be recovered, but may be detected in tissues for the purpose of evaluating the utility of the delivery system.  
       [0046] WO99/35260 describes methods for purification and refolding (where required) of protein products expressed in prokaryotic and eukaryotic systems and its contents are incorporated herein by reference.  
       [0047] Nucleic acid of the invention may encode an additional cysteine residue which can be expressed at the carboxy-terminus or other location within a polypeptide of the invention. The utility and post-translational modification of the carboxy-terminal cysteine is described in WO99/35260.  
       [0048] The use of insect cells infected with recombinant baculovirus encoding the oligomeric polypeptide is a preferred general method for preparing complex proteins, particularly an oligomeric polypeptide of the invention encoding C3d oligomers or fusions of the C3d oligomers with an antigen.  
       [0049] The use of DNA immunization vectors or recombinant live organisms is an alternative general method for delivery of an oligomeric polypeptide encoding C3d oligomers fused to antigen in vivo as an immunogen for prophylactic or therapeutic purposes.  
       GENERAL METHODS USED IN EXAMPLES  
       [0050] (i) DNA Cleavage  
       [0051] Cleavage of DNA by restriction endonucleases was carried out according to the manufacturer&#39;s instructions using supplied buffers (New England Biolabs (U.K.) Ltd., Herts. or Promega Ltd., Hants, UK). Double digests were carried out simultaneously if the buffer conditions were suitable for both enzymes. Otherwise double digests were carried out sequentially where the enzyme requiring the lowest salt condition was added first to the digest. Once the digest was complete the salt concentration was altered and the second enzyme added.  
       [0052] (ii) DNA Ligation  
       [0053] Ligations were carried out using T4 DNA ligase purchased from Promega or New England Biolabs as described in Sambrook et al, (1989) Molecular Cloning: A Laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory Press.  
       [0054] (iii) Plasmid Isolation  
       [0055] Plasmids were isolated using Wizard™ Plus Minipreps (Promega) or Qiex mini or midi kits and Qiagen Plasmid Maxi kit (QIAGEN, Surrey) according to the manufacturer&#39;s instructions.  
       [0056] (iv) DNA Fragment Isolation  
       [0057] DNA fragments were excised from agarose gels and DNA extracted using the QIAEX gel extraction kit or Qiaquick (QIAGEN, Surrey, UK), or GeneClean, or GeneClean Spin Kit or MERmaid Kit, or MERmaid Spin Kit (Bio 101 Inc, CA. USA) gel extraction kits according to the manufacturer&#39;s instructions.  
       [0058] (v) Introduction of DNA Into  E. coli    
       [0059] Plasmids were transformed into competent  E. coli  BL21 (DE3) or XL1-blue strains (Studier and Moffat, (1986), J. Mol. Biol. 189:113). The  E. coli  strains were purchased as a frozen competent cultures from Stratagene (Cambridge, UK).  
       [0060] (vi) DNA Sequencing  
       [0061] The sequences were analysed by a Perkin Elmer ABI Prism 373 DNA Sequencer. This is an electrophoretic technique using 36 cm×0.2 mm 4% acrylamide gels, the fluorescently labeled DNA fragments being detected by a charge coupled device camera according to the manufacturer&#39;s instructions.  
       [0062] (vii) Production of Oligonucleotides and Synthetic Genes  
       [0063] Oligonucleotides and synthetic genes were purchased from Cruachem, Glasgow, UK or from Sigma-Genosys, Cambridge, UK.  
       [0064] (viii) Generation of Baculovirus Vectors  
       [0065] Plasmids described in this invention having the prefix pBP or pBAC are used to generate baculovirus vectors and express the encoded recombinant polypeptides by the following methods (Sections (viii) to (x)). Purified plasmid DNA was used to generate recombinant baculoviruses using the kits ‘The BacPak Baculovirus Expression System’ (Clontech, CA, USA) or ‘BacVector 3000’ (Invitrogen) according to the manufacturers&#39; protocols. The insect cell line Sf9 (ATCC) was grown in Sf900II medium (Gibco) at certain times supplemented with foetal calf serum (Gibco, Paisley, UK). Cells were transfected with the linearised baculovirus DNA (supplied in the kits) and the purified plasmid. Plaque assays (see method below) were carried out on culture supernatants and a series of ten-fold dilutions thereof to allow isolation of single plaques. Plaques were picked using glass Pasteur pipettes and transferred into 0.5 ml aliquots of growth medium. This is the primary seed stock.  
       [0066] (ix) Plaque Assay of Baculoviruses  
       [0067] 1×10 6  Sf9 cells were seeded as monolayer cultures in 30 mm plates and left to attach for at least 30 minutes. The medium was poured off and virus inoculum in 100 μl growth medium was dripped onto the surface of the monolayer. The plates were incubated for 30 minutes at room temperature, occasionally tilting the plates to prevent the monolayer from drying out. The monolayer was overlaid with a mixture of 1 ml growth medium and 3% (w/v) “Seaplaque” agarose (FMC, ME) warmed to 37° C. and gently swirled to mix in the inoculum. Once set a liquid overlay of 1 ml growth medium was applied. The plates were incubated in a humid environment for 3-5 days.  
       [0068] Visualisation of plaques was achieved by addition to the liquid overlay 1 ml phosphate buffered saline (PBS) containing neutral red solution at 0.1% (w/v) from a stock solution of 1% (w/v) (Sigma, Dorset,UK). Plaques were visible as circular regions devoid of stain up to 3 mm in diameter.  
       [0069] (x) Scale-Up of Baculovirus Vectors and Protein Expression  
       [0070] 200 μl of the primary seed stock was used to infect 1×10 6  Sf9 monolayer cell cultures in 30 mm plates. The seed stock was dripped onto the monolayer and incubated for 20 minutes at room temperature, and then overlaid with 1 ml growth medium. The plates were incubated at 27° C. in a humid environment for 3-5 days. The supernatant from these cultures is Passage 1 virus stock. The virus titre was determined by plaque assay and further scale up was achieved by infection of monolayer cultures or suspension cultures at a multiplicity of infection (moi) of 0.1. Virus stock were passaged a maximum of six times to minimise the emergence of defective virus.  
       [0071] Expression of recombinant proteins was achieved by infection of monolayer or suspension cultures in growth medium with or without foetal calf serum (FCS). Where FCS was omitted cells conditioned to growth in the absence of FCS were used. Virus stocks between passage 1 and 6 were used to infect cultures at a moi of &gt;5 per cell. Typically, infected cultures were harvested 72 hours post infection and recombinant proteins isolated either from the supernatants or the cells.  
       [0072] (xiv) Protein Purification  
       [0073] A number of standard chromatographic techniques can be used to isolate the C3d-containing proteins, e.g. such methods as ion-exchange and hydrophobic interaction matrixes chromatography utilising the appropriate buffer systems and gradient to purify the target proteins. The properties of the C3d containing fusion polypeptides will vary depending on the nature of the fusion protein. Examples of methods employed are described in WO99/35260.  
       [0074] (xv) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)  
       [0075] SDS-PAGE was carried out generally using the Novex system (Novex GmbH, Heidleburg) according to the manufacturer&#39;s instructions. Pre-packed gels of Tris/glycine a 4-20% acrylamide gradient were usually used. Samples for electrophoresis, including protein molecular weight standards (for example LMW Kit, Pharmacia, Sweden or Novex Mark 12, Novex, Germany) were usually diluted in 1% (w/v) SDS—containing buffer (with or without 5% (v/v) 2-mercaptoethanol), and left at room temperature for 5 to 30 min before application to the gel.  
       [0076] (xvi) Immunoblotting  
       [0077] (a) Dot Blot  
       [0078] Immobilon membranes (Millipore, Middlesex, UK) were activated by immersion in methanol for 20 seconds and then washed in PBS for five minutes. The membrane was placed into a vacuum manifold Dot Blotter (Bio-Rad Laboratories, Watford, UK). Crude extracts from cells or culture supernatants were transferred onto the membrane by applying a vacuum and washed through with PBS. Without allowing the membrane to dry out, the Dot Blotter was dismantled and the membrane removed.  
       [0079] (b) Western Blotting  
       [0080] Samples of cell extracts and purified proteins were separated on SDS-PAGE as described in Section (xv). The Immobilon membrane was prepared for use as in (a) above. The gel and the membrane were assembled in the Semi-Dry Transfer Cell (Trans-Blot SD, Bio-Rad Laboratories) with the Immobilon membrane towards the anode and the SDS-PAGE gel on the cathode side. Between the cathode and the gel were placed 3 sheets of Whatman 3M filter paper cut to the size of the gel pre-soaked in a solution of 192 mM 6-amino-n-caproic acid, 25 mM Tris pH 9.4 containing 10% (v/v) methanol. Between the anode and the membrane were placed two sheets of Whatman 3M filter paper cut to the size of the gel and soaked in 0.3M -Tris pH 10.4 containing 10% (v/v) methanol next to the anode and on this was laid a further sheet of Whatman 3M filter paper pre-soaked in 25 mM Tris pH 10.4 containing 10% (v/v) methanol.  
       [0081] The whole-assembled gel assembly was constructed to ensure the exclusion of air pockets. The proteins were transferred from the SDS-PAGE to the Immobilon membrane by passing 200 mA current through the assembly for 30 minutes.  
       [0082] (c) Immunoprobing of Dot Blot and Western Membranes  
       [0083] The membranes were blocked by incubating the membrane for 1 h at room temperature in 50 ml of 10 mM phosphate buffer pH 7.4 containing 150 mM NaCl, 0.02% (w/v) Ficoll 400, 0.02% (w/v) polyvinylpyrolidine and 0.1% (w/v) bovine serum albumin (BSA). The appropriate primary antibody was diluted to its working concentration in antibody diluent, 20 mM sodium phosphate buffer pH 7.4 containing 0.3M NaCl, 0.5% (v/v) Tween-80 and 1.0% (w/v) BSA. The membrane was incubated for 2 h at room temperature in 50 ml of this solution and subsequently washed three times for 2 minutes in washing buffer, 20 mM sodium phosphate pH 7.4 containing 0.3M NaCl and 0.5% (v/v) Tween-80. The membrane was then transferred to 50 ml of antibody diluent buffer containing a suitable dilution of the species specific antibody labelled with the appropriate label, e.g. biotin, horse radish peroxidase (HRP), for the development process chosen and incubated for 2 h at room temperature. The membrane was then washed in washing buffer as described above. Finally, the blot was developed according to the manufacturer&#39;s instructions.  
       [0084] The appropriate dilution of antibody for both the primary and secondary antibodies refers to the dilution that minimises unwanted background noise without affecting detection of the chosen antigen using the development system chosen. This dilution is determined empirically for each antibody.  
       [0085] (xvii) Gene Sequences  
       [0086] The sequence of wild-type human C3d is available on public databases under accession number K02765. Other published C3d sequences include mouse  Mus musculus  (K02782), rat  Rattus norvegicus  (X52477), guinea pig  Cavia porcellus  (M34054), rabbit  Oryctolagus cuniculus  (M32434), sheep  Ovis aries  (AF038130), chicken  Gallus gallus  (U16848), cobra  Naja naja  (L02365), lamprey  Lampetra japonica  (D10087) , toad  Xenopus laevis  (U19253), carp  Cyprinus carpio  (AB016210), trout  Oncrhynchus mykiss  (L24433) and sea urchin  Strongylocentrotus purpuratus  (AFO25526).  
       [0087] Variant gene sequences for human and mouse C3d are given in WO99/35260. The sequence of all novel species-specific C3d sequences and variant DNA sequences encoding concatamers of the same polypeptide are described in the following examples and in the appendices. 
     
    
    
     EXAMPLES  
     [0088] 1. Cloning of C3d From Non-Human Vertebrate Tissue Using Degenerate Primers  
     [0089] 1.1 Primer Design  
     [0090] The degenerate primers used to clone the species-specific C3d sequences were designed by alignment of existing C3 protein sequences from human, mouse, rat, and guinea pig. Regions of amino acid conservation within and flanking the C3d region, where low codon redundancy was prominent were selected by eye, and oligonucleotides for RT-PCR designed to incorporate redundant bases where necessary  
                                  (FARM   TGY GGR GAR CAG AAC ATG ATY GGC ATG   SEQID1           1)               (FARM   CCG TAG TAT CTY ASN TCR TTG AGC CA   SEQID2       2)               (FARM   GGA GTC TTC GAG GAG AAT GGG CC   SEQID3       3)               (FARM   GTG TGT CWG GRR CRA AGC CRG TCA TCA T   SEQID4       4)               (FARM   GTR ATG CAG GAC TTC TTC ATY GAC CTG   SEQID5       5)               (FARM   GGC TGT CAG GGA CAC GTC TTT CTC   SEQID6       6)               (FARM   GCA AGG GAC CCC MGT GGC CCA GAT G   SEQID7       7)               (FARM   GYC ACC ACC GAC AAK GTG CCT TG   SEQID8       8)                 R = G/A, Y = C/T, W = A/T, S = G/C, K = G/T,       M = A/C, N = A/C/G/T.          
 
     [0091] 1.2 Reverse Transcription-PCR  
     [0092] Total RNA was purified from liver or other tissue samples of horse ( Equus caballus ), pig ( Sus scrofa ), marmoset (Callithrix sp.), cat ( Felis catus ) and dog ( Canis familiaris ) by the acid-guanidinium thiocyanate-phenol chloroform extraction technique of Chomczynski and Sacchi (Anal. Biochem 162: 156-159 (1987)). RNA extracted from bovine liver ( Bos taurus ) was obtained commercially (Clontech). Approximately 3 ug of RNA was used in the RT reaction using the reverse transcription system from Promega. Reverse transcription was primed with 40 pmol of anti-mRNA sense primer, (ie. any of the even-numbered primers).  
                 
 
     [0093] In some cases a single round PCR was sufficient to generate a positive product, whereas on others nested PCR was necessary. For example an outer PCR with primers FARM 4 and 5 was followed by inner PCR with primers FARM 6 &amp;7 and 3 &amp;8, thus covering the entire C3d region. PCR conditions were typically 95° C. 30 sec, 54° C. 30 sec, 72° C. 60 sec, x35 cycles.  
     [0094] 1.3 Subcloning and Sequencing of Novel C3d Clones From Cow, Pig, Horse and Marmoset, Cat and Dog  
     [0095] PCR products derived from horse ( Equus Caballus ), pig ( Sus scrofa ), marmoset (Callithrix sp.), cat ( Felis catus ) and dog ( Canis familiaris ) and cow ( Bos taurus ) were subcloned into pU57/T (MBI Fermentas) and a minimum of three clones covering any region of C3d were fully sequenced and aligned using the SeqMan module of the DNAStar software package. The amino acid and nucleic acid sequences are given in SEQID 9-18  
     [0096] 1.4 Design of Variant Genes to Prevent Homologous Recombination  
     [0097] For each native sequence published or cloned de novo variant genes may be designed which encode the same amino acid sequence but which contain a large number of silent mutations. These sequences may be cloned in isolation or in tandem with the native sequence and are resistant to homologous recombination. These sequences allow expression of concatamers of C3d from DNA which would otherwise undergo homologous recombination. In addition when used in DNA immunization vectors, or in vectors derived from live organisms with the intention of raising antibodies to antigens cloned in tandem to the C3d, the variant genes are resistant to homologous recombination with the native C3d present in the genome of the host species. Examples of variant genes for pig, cow and dog are given in SEQID 19 to 24.  
     Appendix 1  
     Sequences Described in the Text  
     [0098] SEQID1: Nucleotide sequence of PCR primer FARM 1  
     [0099] TGYGGRGARCAGAACATGATYGGCATG  
     [0100] SEQID2: Nucleotide sequence of PCR primer FARM 2  
     [0101] CCGTAGTATCTYASNTCRTTGAGCCA  
     [0102] SEQID3: Nucleotide sequence of PCR primer FARM 3  
     [0103] GGAGTCTTCGAGGAGAATGGGCC  
     [0104] SEQID4: Nucleotide sequence of PCR primer FARM 4  
     [0105] GTGTGTCWGGRRCRAAGCCRGTCATCAT  
     [0106] SEQID5: Nucleotide sequence of PCR primer FARM 5  
     [0107] GTRATGCAGGACTTCTTCATYGACCTG  
     [0108] SEQID6: Nucleotide sequence of PCR primer FARM 6  
     [0109] GGCTGTCAGGGACACGTCTTTCTC  
     [0110] SEQID7: Nucleotide sequence of PCR primer FARM 7  
     [0111] GCA AGG GAC CCC MGT GGC CCA GAT G  
     [0112] SEQID8: Nucleotide sequence of PCR primer FARM 8  
     [0113] GYC ACC ACC GAC AAK GTG CCT TG  
     [0114] R=G/A, Y=C/T, W=A/T, S=G/C, K=G/T, M=A/C, N=A/C/G/T.  
     [0115] SEQID9: Amino acid sequence of C3d from cow  
                              1   His Leu Ile Gln Thr Pro Ser Gly Cys Gly                   11   Glu Gln Asn Met Ile Gly Met Thr Pro Thr               21   Val Ile Ala Val His Tyr Leu Asp Ser Thr               31   Asp Gln Trp Glu Lys Phe Gly Leu Glu Lys               41   Arg Gln Glu Ser Leu Glu Leu Ile Arg Lys               51   Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln               61   Lys Ser Ser Ala Tyr Ala Ala Phe Gln Tyr               71   Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr               81   Val Val Lys Val Phe Ala Leu Ala Ala Asn               91   Leu Ile Ala Ile Asp Ser Lys Asp Leu Cys               101   Glu Thr Val Lys Trp Leu Ile Leu Glu Lys               111   Gln Lys Pro Asp Gly Ile Phe Gln Glu Asp               121   Gly Pro Val Ile His Gln Glu Met Ile Gly               131   Gly Phe Arg Asp Thr Arg Glu Lys Asp Val               141   Ser Leu Thr Ala Phe Val Leu Ile Ala Leu               151   His Glu Ala Lys Asp Ile Cys Glu Ala Gln               161   Val Asn Ser Leu Gly Arg Ser Ile Ala Lys               171   Ala Gly Asp Phe Leu Glu Asn His Tyr Arg               181   Glu Leu Arg Arg Pro Tyr Thr Val Ala Ile               191   Ala Ala Tyr Ala Leu Ala Leu Leu Gly Lys               201   Leu Glu Gly Asp Arg Leu Thr Lys Phe Leu               211   Asn Thr Ala Lys Glu Lys Asn Arg Trp Glu               221   Glu Pro Asn Gln Lys Leu Tyr Asn Val Glu               231   Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu               241   Ala Arg Lys Asp Tyr Asp Thr Thr Pro Pro               251   Val Val Arg Trp Leu Asn Glu Gln Arg Tyr               261   Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala               271   Thr Phe Met Val Phe Gln Ala Leu Ala Gln               281   Tyr Gln Lys Asp Val Pro Asp His Lys Glu               291   Leu Asn Leu Asp Val Ser Ile Gln Leu Pro          
 
     [0116] SEQID10: Nucleotide sequence of cow C3d  
                                  1   CACCTTATCC AAACCCCCTC CGGCTGTGGG GAGCAGAACA TGATTGGTAT   50                   51   GACGCCCACG GTCATCGCCG TGCACTACCT GGACAGCACC GACCAGTGGG   100               101   AGAAGTTCGG CTTGGAGAAG CGGCAGGAGT CCCTGGAGCT CATCAGAAAG   150               151   GGGTACACCC AGCAGCTGGC CTTCAGACAA AAAAGCTCAG CCTACGCCGC   200               201   CTTCCAATAT CGGCCCCCCA GCACCTGGCT GACAGCCTAC GTGGTCAAGG   250               251   TCTTTGCACT GGCCGCCAAC CTCATCGCCA TAGACTCCAA GGACCTCTGT   300               301   GAGACCGTCA AATGGCTGAT CCTGGAGAAG CAGAAGCCTG ATGGAATCTT   350               351   CCAGGAGGAT GGGCCTGTGA TACACCAAGA AATGATTGGT GGCTTCAGGG   400               401   ACACCAGGGA GAAAGATGTG TCCCTTACAG CCTTTGTTCT CATCGCGCTG   450               451   CACGAGGCTA AAGACATTTG CGAGGCACAG GTCAACAGCC TGGGCCGCAG   500               501   CATCGCTAAG GCAGGAGACT TCCTCGAAAA CCACTACAGA GAGTTGCGAA   550               551   GACCATATAC TGTGGCCATT GCTGCCTATG CCCTGGCTTT GTTGGGCAAG   600               601   CTGGAGGGTG ACCGCCTCAC CAAATTTCTG AACACAGCCA AAGAAAAGAA   650               651   CCGCTGGGAG GAACCCAACC AGAAGCTCTA CAATGTGGAG GCCACGTCCT   700               701   ACGCCCTCTT GGCTCTGCTG GCACGCAAAG ACTACGACAC TACGCCTCCT   750               751   GTCGTGCGCT GGCTCAACGA GCAGAGATAC TATGGAGGTG GTTATGGCTC   800               801   CACGCAGGCC ACTTTCATGG TGTTCCAAGC CTTGGCCCAA TACCAGAAGG   850               851   ATGTTCCTGA TCACAAGGAG CTGAACCTGG ATGTGTCCAT CCAACTGCCC   900          
 
     [0117] SEQID11: Amino acid sequence of C3d from horse  
                              1   His Leu Ile Val Thr Pro Ser Gly Cys Gly                   11   Glu Gln Asn Met Ile Ser Met Thr Pro Thr               21   Val Ile Ala Val His Tyr Leu Asp Gln Thr               31   Glu Gln Trp Glu Lys Phe Gly Leu Glu Lys               41   Arg Gln Glu Ser Leu Glu Leu Ile Lys Lys               51   Gly Tyr Thr Gln Gln Leu Ala Tyr Arg Gln               61   Pro Ser Ser Ala Tyr Ala Ala Phe Leu Ser               71   Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr               81   Val Val Lys Val Phe Ala Leu Ala Ser Asn               91   Leu Ile Ala Ile Asp Ser Gln Val Leu Cys               101   Gly Ala Val Lys Trp Leu Ile Leu Gln Lys               111   Gln Lys Pro Asp Gly Val Phe Gln Glu Asp               121   Gly Pro Val Ile His Gln Glu Met Ile Gly               131   Gly Phe Arg Asn Ala Glu Glu Lys Asp Val               141   Ser Leu Thr Ala Phe Val Leu Ile Ala Leu               151   Gln Glu Ala Lys Asp Ile Cys Glu Gly Gln               161   Val Asn Ser Leu Ala Arg Ser Ile Ile Lys               171   Ala Gly Asp Phe Leu Glu Ala His Tyr Asn               181   Asn Leu Arg Arg Pro Tyr Ser Val Ala Ile               191   Ala Gly Tyr Ala Leu Ala Gln Met Gly Lys               201   Leu Glu Asp Pro Leu Leu Asn Lys Phe Leu               211   Ser Ala Ala Thr Asp Arg Asn Arg Trp Glu               221   Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu               231   Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu               241   Leu Leu Arg Asp Phe Asp Ser Val Pro Pro               251   Val Val Arg Trp Leu Asn Glu Gln Arg Tyr               261   Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala               271   Thr Phe Met Val Phe Gln Ala Leu Ala Gln               281   Tyr Gln Lys Asp Val Pro Asn His Lys Asp               291   Leu Asn Leu Asp Val Ser Ile Asn Leu Pro          
 
     [0118] SEQID12: Nucleotide sequence of horse C3d  
                                  1   CACCTCATCG TGACGCCCTC GGGCTGCGGC GAGCAGAACA TGATTAGCAT   50                   51   GACGCCCACG GTCATCGCAG TGCATTACCT GGACCAGACC GAGCAGTGGG   100               101   AGAAGTTCGG CCTGGAGAAG CGGCAGGAGT CCTTGGAGCT CATCAAGAAG   150               151   GGGTACACCC AGCAGCTGGC CTACAGACAA CCCAGCTCAG CCTATGCAGC   200               201   CTTCCTGAGC CGGCCGCCCA GCACCTGGCT GACAGCCTAC GTGGTCAAGG   250               251   TCTTCGCTCT GGCCTCCAAC CTCATCGCCA TCGACTCCCA GGTCCTCTGT   300               301   GGGGCTGTCA AATGGCTGAT CCTCCAGAAG CAGAAGCCAG ATGGAGTCTT   350               351   CCAGGAGGAC GGGCCCGTGA TACATCAAGA AATGATTGGT GGCTTCCGGA   400               401   ATGCGGAGGA GAAAGACGTG TCCCTCACAG CCTTTGTTCT CATCGCACTG   450               451   CAGGAAGCTA AAGATATTTG CGAGGGACAG GTCAACAGCC TGGCACGCAG   500               501   CATCATTAAG GCAGGAGACT TCCTTGAAGC CCACTATAAT AACCTGCGGA   550               551   GACCATATTC TGTGGCCATT GCTGGCTACG CCCTGGCCCA GATGGGCAAG   600               601   CTGGAGGACC CCCTCCTCAA CAAATTCCTG AGCGCAGCCA CAGACAGGAA   650               651   CCGCTGGGAG GAGCCTGGCC AGAAGCTCTA CAATGTAGAG GCCACATCCT   700               701   ACGCCCTCTT GGCCCTGCTG CTGCTCAGAG ACTTTGACTC TGTGCCTCCG   750               751   GTGGTGCGCT GGCTCAACGA ACAGAGATAC TACGGAGGTG GCTATGGCTC   800               801   CACCCAGGCC ACCTTCATGG TGTTCCAAGC CTTGGCTCAG TACCAAAAGG   850               851   ATGTCCCTAA CCACAAGGAC CTGAACCTCG ATGTTTCCAT CAACCTGCCC   900          
 
     [0119] SEQID13: Amino acid sequence of C3d from marmoset  
                              1   His Leu Ile Val Thr Pro Ser Gly Cys Gly                   11   Glu Gln Asn Met Ile Gly Met Thr Pro Thr               21   Val Ile Ala Val His Tyr Leu Asp Gln Thr               31   Glu Gln Trp Glu Lys Phe Gly Leu Glu Lys               41   Arg Gln Gly Ala Val Glu Leu Ile Lys Lys               51   Gly Tyr Ala Gln Gln Leu Ala Phe Lys Gln               61   Pro Ser Ser Ala Tyr Ala Ala Phe Leu Asn               71   Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr               81   Val Val Lys Val Phe Ser Leu Ala Val Asn               91   Leu Ile Ala Ile Asp Ser Gln Val Leu Cys               101   Gly Ala Val Lys Trp Leu Ile Leu Glu Lys               111   Gln Lys Pro Asp Gly Val Phe Gln Glu Asp               121   Gly Pro Val Ile His Gln Glu Met Ile Gly               131   Gly Phe Arg Asn Thr Gln Glu Lys Asp Met               142   Ala Leu Thr Ala Phe Val Leu Ile Ser Leu               151   Gln Glu Ala Lys Asp Ile Cys Glu Glu Leu               161   Val Asn Ser Leu Pro Arg Ser Ile Ile Asp               171   Ala Gly Asn Phe Leu Glu Ala Asn Tyr Met               181   Asn Leu Gln Arg Ser Tyr Thr Val Ala Ile               191   Ala Gly Tyr Ala Leu Ala Gln Leu Asp Lys               201   Leu Asn Gly Pro Leu Leu Asn Lys Phe Leu               211   Ser Thr Ala Lys Asp Lys Asn Arg Trp Glu               221   Glu Pro Gly Gln Gln Leu Tyr Asn Val Glu               231   Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu               241   Gln Met Lys Asp Phe Asp Phe Val Pro Pro               251   Val Val Arg Trp Leu Asn Glu Gln Arg Tyr               261   Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala               271   Thr Phe Met Val Phe Gln Ala Leu Ala Gln               281   Tyr Gln Lys Asp Val Pro Asp His Lys Glu               291   Leu Asn Leu Asp Val Ser Leu Gln Leu Pro          
 
     [0120] SEQID14: Nucleotide sequence of marmoset C3d  
                                  1   CACCTCATCG TGACCCCCTC GGGCTGTGGG GAACAGAACA TGATTGGCAT   50                   51   GACACCCACG GTCATCGCGG TGCATTACCT GGATCAAACG GAGCAGTGGG   100               101   AGAAGTTCGG CTTGGAGAAG CGGCAGGGAG CCGTGGAGCT CATCAAGAAG   150               151   GGGTACGCAC AGCAGCTGGC CTTCAAACAA CCCAGCTCTG CCTATGCGGC   200               201   CTTCCTGAAC CGGCCACCCA GCACCTGGCT GACCGCCTAC GTGGTCAAGG   250               251   TCTTCTCTCT GGCCGTCAAC CTCATTGCCA TTGACTCCCA AGTCCTCTGT   300               301   GGGGCTGTTA AATGGCTGAT CCTGGAGAAG CAGAAGCCTG ATGGGGTCTT   350               351   CCAGGAGGAT GGGCCCGTGA TACACCAAGA AATGATTGGT GGCTTCCGGA   400               401   ACACCCAGGA GAAAGACATG GCCCTCACGG CCTTTGTTCT CATCTCGCTG   450               451   CAGGAGGCTA AAGATATTTG CGAGGAGCTT GTCAACAGCC TGCCACGCAG   500               501   CATCATTGAT GCAGGAAACT TCCTTGAAGC CAACTACATG AACCTACAGA   550               551   GATCCTACAC TGTGGCCATC GCTGGCTATG CCCTGGCCCA GCTGGACAAA   600               601   CTGAACGGGC CTCTTCTCAA CAAATTTCTG AGCACAGCCA AAGATAAGAA   650               651   CCGCTGGGAG GAGCCTGGTC AGCAGCTCTA CAACGTGGAG GCCACATCCT   700               701   ATGCCCTCTT GGCCCTCCTG CAGATGAAAG ACTTCGACTT TGTGCCTCCC   750               751   GTCGTGCGTT GGCTCAATGA GCAGAGATAC TACGGTGGCG GCTATGGCTC   800               801   TACCCAGGCC ACCTTCATGG TGTTCCAAGC CTTGGCCCAA TACCAAAAGG   850               851   ACGTCCCTGA CCACAAGGAA CTGAACCTGG ATGTGTCCCT CCAACTGCCA   900          
 
     [0121] SEQID 15: Amino acid sequence of C3d from pig  
                              1   His Leu Ile Gln Thr Pro Ser Gly Cys Gly                   11   Glu Gln Asn Met Ile Gly Met Thr Pro Thr               21   Val Ile Ala Val His Tyr Leu Asp Ser Thr               31   Glu Gln Trp Glu Lys Phe Gly Leu Glu Lys               41   Arg Gln Glu Ala Leu Glu Leu Ile Lys Lys               51   Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln               61   Lys Asn Ser Ala Phe Ala Ala Phe Gln Asp               71   Arg Leu Ser Ser Thr Trp Leu Thr Ala Tyr               81   Val Val Lys Val Phe Ala Met Ala Ala Asn               91   Leu Ile Ala Ile Asp Ser Gln Val Leu Cys               101   Gly Ala Val Lys Trp Leu Ile Leu Glu Lys               111   Gln Lys Pro Asp Gly Val Phe Glu Glu Asn               121   Gly Pro Val Ile His Gln Glu Met Ile Gly               131   Gly Phe Lys Asn Thr Glu Glu Lys Asp Val               141   Ser Leu Thr Ala Phe Val Leu Ile Ala Leu               151   Gln Glu Ala Lys Asp Ile Cys Glu Pro Gln               161   Val Asn Ser Leu Leu Arg Ser Ile Asn Lys               171   Ala Arg Asp Phe Leu Ala Asp Tyr Tyr Leu               181   Glu Leu Lys Arg Pro Tyr Thr Val Ala Ile               191   Ala Gly Tyr Ala Leu Ala Leu Ser Asp Lys               201   Leu Asp Glu Pro Phe Leu Asn Lys Leu Leu               211   Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu               221   Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu               231   Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu               241   Val Val Lys Asp Phe Asp Ser Val Pro Pro               251   Ile Val Arg Trp Leu Asn Glu Gln Arg Tyr               261   Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala               271   Thr Phe Met Val Phe Gln Ala Leu Ala Gln               281   Tyr Gln Lys Asp Val Pro Asp His Lys Asp               291   Leu Asn Leu Asp Val Ser Ile His Leu Pro          
 
     [0122] SEQID16: Nucleotide sequence of pig C3d  
                                  1   CACCTCATCC AAACCCCCTC CGGCTGTGGG GAGCAGAACA TGATCGGCAT   50                   51   GACGCCCACA GTCATCGCTG TGCACTACCT GGACAGCACC GAACAATGGG   100               101   AGAAGTTCGG CCTGGAGAAG AGGCAGGAAG CCTTGGAGCT CATCAAGAAG   150               151   GGGTACACCC AGCAACTGGC CTTCAGACAA AAGAACTCAG CCTTTGCCGC   200               201   CTTCCAGGAC CGGCTGTCCA GCACCTGGCT GACAGCCTAT GTGGTCAAGG   250               251   TCTTCGCTAT GGCAGCCAAC CTCATCGCCA TCGACTCCCA GGTCCTCTGT   300               301   GGGGCCGTCA AATGGCTGAT CCTGGAGAAG CAGAAGCCTG ATGGAGTCTT   350               351   CGAGGAGAAT GGGCCCGTGA TACACCAAGA AATGATTGGT GGCTTCAAGA   400               401   ACACTGAGGA GAAAGACGTG TCCCTGACAG CCTTTGTTCT CATCGCGCTG   450               451   CAGGAGGCTA AAGACATCTG TGAACCACAG GTCAATAGCC TGTTGCGCAG   500               501   CATCAATAAG GCAAGAGACT TCCTCGCAGA CTACTACCTA GAATTAAAAA   550               551   GACCATATAC TGTGGCCATT GCTGGTTATG CCCTGGCTCT ATCTGACAAG   600               601   CTGGATGAGC CCTTCCTCAA CAAACTTCTG AGCACAGCCA AAGAAAGGAA   650               651   CCGCTGGGAG GAACCTGGCC AGAAGCTCTA CAATGTGGAG GCCACATCCT   700               701   ACGCCCTCTT GGCTCTGCTG GTAGTCAAAG ACTTTGACTC TGTCCCTCCT   750               751   ATTGTGCGCT GGCTCAATGA GCAGAGATAC TACGGAGGTG GCTACGGATC   800               801   TACCCAGGCC ACTTTCATGG TGTTCCAAGC CTTGGCCCAA TACCAGAAGG   850               851   ATGTCCCTGA TCACAAGGAT CTGAACCTGG ATGTGTCCAT CCACCTGCCC   900          
 
     [0123] SEQID 17: Amino acid sequence of C3d from dog  
                              1   His Leu Ile Val Thr Pro Ser Gly Cys Gly                   11   Gln Glu Asn Met Ile Gly Met Thr Pro Thr               21   Val Ile Ala Leu His Tyr Leu Asp Glu Thr               31   Gln Glu Trp Asp Lys Phe Gly Leu Gln Lys               41   Arg Glu Gln Ala Leu Gln Leu Ile Lys Lys               51   Gly Tyr Thr Glu Glu Leu Ala Phe Arg Glu               61   Pro Asn Ser Ala Phe Ala Ala Phe Glu Asn               71   Arg Pro Ser Ser Thr Trp Leu Thr Ala Tyr               81   Val Val Lys Val Phe Ser Leu Ala Thr Asn               91   Leu Ile Ala Ile Gln Ala Glu Val Leu Cys               101   Gly Ala Val Lys Trp Leu Ile Leu Gln Lys               111   Glu Lys Pro Asp Gly Ile Phe Glu Gln Asp               121   Gly Pro Val Ile His Gln Glu Met Thr Gly               131   Gly Phe Arg Glu Ala Glu Glu Lys Ser Val               141   Ser Leu Thr Ala Phe Val Leu Ile Ala Leu               151   Lys Glu Ala Glu Asp Ile Cys Ile Gly Gln               161   Val Asn Ile Leu Pro Ser Ser Ile Glu Lys               171   Ala Gly Asn Tyr Leu Ala Ala His Tyr Gln               181   Asn Leu Arg Arg Pro Tyr Ser Val Ala Ile               191   Ala Gly Tyr Ala Leu Ala His Leu Asp Lys               201   Leu Glu Gly Asp Asn Leu Arg Lys Phe Leu               211   Asn Thr Ala Arg Asp Arg Asn Arg Trp Val               221   Glu Pro Gly Lys Lys Leu Tyr Asn Val Glu               231   Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu               241   Leu Leu Lys Asp Phe Asp Asn Val Pro Pro               251   Val Val Arg Trp Leu Asn Gln Glu Arg Tyr               261   Tyr Gly Gly Gly Tyr Gly Ser Thr Glu Ala               271   Thr Phe Met Val Phe Glu Ala Leu Ala Glu               281   Tyr Glu Lys Asp Val Pro Asn His Lys Asp               291   Leu Asn Leu Glu Val Ser Ile Asn Leu Pro               301   Ser Arg Ser Thr Gln Ile Lys His His Ile               311   Val Trp          
 
     [0124] SEQID 18: Nucleotide sequence of dog C3d  
                                  1   CACCTCATCG TGACCCCGTC GGGCTGCGGG GAGCAGAACA TGATCGGCAT   50                   51   GACGCCCACC GTCATCGCCC TGCATTACCT GGACCAAACC GAGCAGTGGG   100               101   ACAAGTTCGG GCTGGAGAAG CGCCAGGAGG CCTTGGAGCT CATCAAGAAG   150               151   GGATACACCC AACAGCTGGC CTTCAGACAA CCCAACTCGG CCTTCGCTGC   200               201   CTTCCAGAAC CGGCCATCCA GCACCTGGCT GACAGCCTAC GTGGTCAAGG   250               251   TCTTCTCTCT GGCCACCAAC CTCATCGCCA TTGAAGCCCA GGTTCTCTGC   300               301   GGGGCTGTCA AATGGCTGAT CCTGGAGAAG CAGAAGCCCG ATGGGATCTT   350               351   CCAGGAGGAT GGGCCTGTGA TCCACCAAGA GATGACCGGT GGCTTCCGGG   400               401   AAGCTGAGGA GAAGTCTGTG TCCCTCACGG CCTTTGTTCT CATAGCACTA   450               451   AAAGAGGCTG AAGATATTTG TATAGGACAG GTTAACATCT TGCCAAGCAG   500               501   CATCGAAAAG GCAGGAAATT ATCTTGCAGC CCACTACCAG AACCTGAGGA   550               551   GACCATATTC TGTGGCCATT GCTGGCTACG CCCTGGCCCA TCTAGACAAA   600               601   CTGGAGGGAG ATAACCTCAG AAAATTTCTG AACACAGCCA GAGACAGGAA   650               651   CCGCTGGGTG GAGCCTGGCA AGAAGCTCTA CAATGTGGAA GCCACATCCT   700               701   ACGCCCTCTT GGCCCTGCTG CTGCTCAAAG ACTTTGACAA TGTACCTCCT   750               751   GTCGTCCGCT GGCTCAATGA GCAGAGATAC TACGGAGGTG GCTATGGCTC   800               801   CACCCAGGCC ACCTTCATGG TGTTCCAAGC CTTGGCCCAA TACCAGAAGG   850               851   ATGTCCCCAA CCACAAGGAC CTGAACCTGC AAGTGTCCAT CAACCTGCCC   900               901   AGCCGCAGCA CAGAGATCAA GCACCACATT GTCTGG 936          
 
     [0125] SEQID19: Nucleotide sequence of first variant cow C3d  
                                  1   CACCTGATTC AAACTCCTAG CGGGTGCGGC GAGCAAAACA TGATTGGGAT   50                   51   GACCCCTACA GTTATCGCAG TCCACTACCT CGATTCCACT GACCAATGGG   100               101   AAAAATTCGG ACTGGAAAAA CGCCAAGAGA GCCTCGAGTT GATTAGGAAA   150               151   GGCTACACTC AGCAACTCGC ATTCCGTCAG AAATCTTCCG CTTACGCGGC   200               201   TTTCCAGTAT AGGCCTCCTT CCACATGGCT CACTGCATAC GTCGTGAAAG   250               251   TGTTTGCCTT GGCTGCTAAC TTGATCGCTA TTGACAGCAA AGACCTGTGT   300               301   GAAACGGTGA AGTGGCTCAT TCTCGAAAAG CAAAAGCCAG ATGGTATTTT   350               351   TCAAGAGGAC GGCCCAGTCA TTCACCAGGA AATGATCGGC GGTTTTCGCG   400               401   ACACTAGAGA GAAGGATGTC AGTCTCACCG CTTTTGTGCT GATTGCCCTC   450               451   CACGAAGCAA AAGATATCTG CGAAGCTCAG GTGAATTCTC TCGGGCGTAG   500               501   TATCGCCAAG GCTGGTGATT TCTTGGAGAA CCACTACCGT GAGCTCCGCA   550               551   GGCCATATAC CGTCGCTATT GCCGCTTATG CACTCGCCCT CCTGGGGAAG   600               601   TTAGAGGGAG ATCGTCTGAC TAAATTCCTC AACACGGCAA AAGAGAAGAA   650               651   TCGCTGGGAA GAACCTAACC AAAAGTTGTA TAATGTTGAG GCAACCAGCT   700               701   ACGCACTGCT GGCACTCCTC GCTAGGAAGG ACTATGACAC CACTCCTCCA   750               751   GTGGTCCGTT GGTTGAACGA ACAGCGCTAC TACGGTGGCG GCTATGGGTC   800               801   TACCCAGGCT ACATTTATGG TCTTCCAGGC TCTGGCTCAA TATCAGAAAG   850               851   ATGTCCCAGA TCACAAGGAA CTCAATCTCG ATGTTTCTAT CCAATTGCCT   900          
 
     [0126] SEQID 20: Nucleotide sequence of second variant cow C3d  
                                  1   CACTTGATCC AGACACCATC TGGTTGTGGA GAACAAAATA TGATCGGCAT   50                   51   GACACCAACC GTGATTGCTG TTCACTATTT GGATAGTACA GATCAGTGGG   100               101   AAAAGTTTGG GCTCGAGAAA AGACAGGAAT CTCTTGAACT GATCCGCAAA   150               151   GGATATACAC AACAGTTGGC TTTTCGCCAA AAGTCCAGCG CATATGCAGC   200               201   ATTTCAATAC CGCCCACCAT CTACTTGGTT GACCGCTTAT GTTGTTAAGG   250               251   TTTTCGCTCT CGCAGCAAAT CTGATTGCAA TCGATTCTAA GGATTTGTGC   300               301   GAGACTGTTA AATGGTTAAT CTTGGAGAAA CAGAAACCTG ACGGGATCTT   350               351   TCAGGAAGAT GGTCCTGTTA TCCACCAGGA GATGATCGGG GGATTTAGAG   400               401   ATACACGTGA AAAAGACGTT TCTCTGACTG CATTCGTCTT GATCGCTTTG   450               451   CACGAGGCCA AGGACATCTG TGAGGCCCAA GTTAATAGTT TGGGTAGATC   500               501   CATTGCAAAA GCCGGGGACT TTCTGGAAAA TCACTATAGG GAACTGAGAC   550               551   GCCCTTACAC AGTAGCAATC GCAGCATACG CTTTGGCACT GCTCGGAAAA   600               601   CTCGAAGGGG ACAGATTAAC AAAGTTTTTG AATACCGCTA AGGAAAAAAA   650               651   CAGATGGGAA GAGCCAAATC AGAAACTGTA CAACGTAGAA GCTACTAGTT   700               701   ATGCTTTGCT CGCCTTGTTG GCCAGAAAAG ATTACGATAC AACCCCACCT   750               751   GTAGTAAGAT GGCTGAATGA GCAAAGGTAT TACGGGGGAG GATACGGAAG   800               801   CACTCAAGCA ACCTTCATGG TTTTTCAAGC ACTCGCACAG TACCAAAAGG   850               851   ACGTTCCTGA CCACAAAGAG TTGAACTTGG ACGTCAGCAT TCAGCTCCCA   900          
 
     [0127] SEQID21: Nucleotide sequence of first variant pig C3d  
                                  1   CACCTGATTC AAACGCCATC AGGGTGCGGA GAGCAAAACA TGATTGGTAT   50                   51   GACCCCAACG GTGATCGCAG TCCACTATCT CGATTCAACG GAACAGTGGG   100               101   AGAAATTCGG ATTAGAGAAA AGACAAGAAG CGCTCGAATT GATCAAAAAG   150               151   GGCTATACGC AGCAGTTAGC TTTTAGACAG AAGAATTCCG CGTTCGCTGC   200               201   GTTCCAAGAC AGACTTAGTT CAACATGGTT AACTGCGTAT GTTGTGAAAG   250               251   TTTTCGCCAT GGCTGCGAAT CTGATTGCGA TCGATTCACA AGTGTTATGT   300               301   GGCGCTGTGA AATGGTTAAT TCTTGAAAAG CAAAAGCCAG ATGGCGTGTT   350               351   TGAGGAAAAT GGCCCAGTCA TTCACCAGGA AATGATCGGC GGATTTAAGA   400               401   ATACGGAGGA AAAAGATGTA TCATTAACGG CATTTGTCTT AATTGCACTT   450               451   CAAGAGGCGA AGGACATTTG TGAGCCTCAG GTGAACAGTT TATTAAGAAG   500               501   TATTAACAAG GCGCGTGACT TTCTGGCGGA TTACTATCTC GAGTTGAAGA   550               551   GGCCTTATAC GGTAGCTATC GCGGGATACG CACTCGCCTT GTCAGATAAG   600               601   TTAGATGAAC CATTTCTGAA TAAATTACTT TCAACGGCAA AAGAGAGAAA   650               651   TCGTTGGGAA GAACCTGGAC AAAAGTTATA TAACGTAGAG GCAACGAGTT   700               701   ACGCACTTCT TGCCCTCTTA GTGGTTAAGG ACTTCGACAG CGTACCTCCA   750               751   ATTGTCAGAT GGCTGAACGA GCAACGCTAC TATGGGGGAG GGTACGGCAG   800               801   CACGCAGGCA ACGTTTATGG TCTTCCAGGC GTTAGCGCAG TACCAAAAGG   850               851   ACGTACCAGA TCACAAAGAT TTAAACCTCG ACGTCAGTAT CCACTTACCA   900          
 
     [0128] SEQID22: Nucleotide sequence of second variant pig C3d  
                                  1   CACTTAATCC AGACTCCTAG TGGATGTGGC GAACAGATTA TGATCGGGAT   50                   51   GACACCTACC GTAATTGCGG TTCACTACTT AGACAGTACA GAGCAATGGG   100               101   AAAAGTTTGG GCTCGAAAAG CGCCAGGAGG CTCTTGAGTT AATTAAGAAA   150               151   GGTTACACAC AACAACTCGC ATTCAGGCAA AAAAACAGTG CATTTGCGGC   200               201   ATTTCAGGAT CGCTTAAGCA GTACGTGGCT CACCGCATAC GTCGTAAAGG   250               251   TGTTTGCGAT GGCCGCAAAC TTAATCGCAA TTGACAGTCA GGTACTGTGC   300               301   GGAGCGGTTA AGTGGCTTAT CTTAGAGAAA CAGAAACCTG ACGGGGTATT   350               351   CGAAGAGAAC GGTCCTGTTA TCCACCAAGA GATGATTGGG GGTTTCAAAA   400               401   ACACCGAAGA GAAGGACGTT AGTCTCACCG CTTTCGTGCT GATCGCCTTA   450               451   CAGGAAGCCA AAGATATCTG CGAACCACAA GTAAATTCAC TCCTTCGTTC   500               501   AATCAATAAA GCCAGGGATT TCTTAGCCGA CTATTACTTG GAACTCAAAC   550               551   GTCCATACAC CGTTGCGATT GCCGGGTATG CTTTAGCGCT CAGCGACAAA   600               601   CTCGACGAGC CTTTCTTAAA CAAGCTGTTA AGTACCGCTA AGGAACGCAA   650               651   CAGGTGGGAG GAGCCAGGTC AGAAACTTTA CAATGTTGAA GCTACCTCAT   700               701   ATGCTTTATT AGCGTTACTC GTCGTAAAAG ATTTTGATTC AGTGCCACCT   750               751   ATCGTACGTT GGTTAAATGA ACAGAGGTAT TACGGTGGCG GATATGGGTC   800               801   AACACAAGCG ACCTTCATGG TATTTCAAGC ACTCGCACAA TATCAGAAAG   850               851   ATGTGCCTGA CCACAAGGAC CTGAATTTAG ATGTATCAAT TCACCTTCCT   900          
 
     [0129] SEQID23: Nucleotide sequence of first variant dog C3d  
                                  1   CACTTAATCG TCACTCCAAG TGGATGCGGC GAACAGAATA TGATCGGAAT   50                   51   GACACCAACG GTAATTGCGC TCCACTATTT AGACCAGACT GAGCAATGGG   100               101   ATAAGTTTGG ACTCGAAAAG AGGCAGGAAG CGCTCGAGTT AATCAAAAAG   150               151   GGCTATACGC AGCAGTTAGC GTTTAGGCAG CCAAATTCCG CGTTTGCAGC   200               201   GTTCCAAAAC AGACCATCAT CAACGTGGCT TACGGCTTAT GTCGTTAAAG   250               251   TGTTCTCATT AGCGACTAAT CTTATTGCAA TCGAGGCTCA GGTCCTTTGT   300               301   GGCGCGGTTA AATGGTTAAT TCTCGAAAAG CAAAAACCAG ACGGCATTTT   350               351   CCAAGAGGAC GGCCCTGTAA TTCACCAAGA AATGACGGGC GGTTTTCGCG   400               401   AAGCGGAAGA GAAATCAGTA AGTCTTACCG CGTTCGTGCT GATTGCGTTG   450               451   AAAGAAGCCG AAGACPTTTG CATTGGGCAG GTCAATATCC TCCCTAGTTC   500               501   AATTGAAAAA GCTGGCAACT ATCTGGCCGC ACACTATCAG AATCTCCGTA   550               551   GGCCTTATAG CGTCGCAATA GCCGGTTACG CATTAGGACA CCTCGATAAA   600               601   TTAGAGGGTG ACAACTTACG CAAGTTTCTC AATACGGCAA GGGATAGAAA   650               651   TCGTTGGGTA GAACCAGGGA AGAAACTTTA TAATGTAGAG GCGACAAGTT   700               701   ACGCGCTGCT CGCTCTTTTA TTACTAAAGG ACTTCGACAA CGTCCCACCT   750               751   GTGGTGAGGT GGCTTAATGA ACAGCGTTAC TATGGCGGAG GATACGGATC   800               801   AACGCAAGCT ACATTCATGG TCTTTCAAGC GCTCGCGCAA TATCAGAAAG   850               851   ATGTGCCAAA TCACAAGGAT CTCAATTTAC AAGTAAGTAT CAATCTCCCA   900          
 
     [0130] SEQID24: Nucleotide sequence of second variant dog C3d  
                                  1   CACCTGATTG TAACGCCTAG CGGTTGTGGA GAGCAAAACA TGATTGGGAT   50                   51   GACCCCTACT GTGATCGCAT TACACTACCT CGATCAAACA GAACAGTGGG   100               101   ACAAATTCGG TTTAGAGAAA CGTCAAGAGG CTTTAGAACT GATTAAGAAA   150               151   GGTTACACCC AACAACTCGC TTTCCGTCAA CCTAACAGTG CTTTCGCGGC   200               201   TTTTCAGAAT CGTCCTAGTA GTACATGGTT AACCGCATAC GTAGTGAAGG   250               251   TATTTAGTCT TGCAACGAAC TTAATCGCTA TCGAAGCGCA AGTGTTGTGC   300               301   GGAGCCGTGA AGTGGCTCAT CTTAGAGAAA CAGAAGCCTG ATGGTATCTT   350               351   TCAGGAAGAT GGACCAGTCA TCCACCAGGA GATGACTGGA GGGTTCAGAG   400               401   AGGCCGAGGA AAAGAGCGTG TCATTGACAG CTTTTGTCTT AATCGCCCTC   450               451   AAGGAGGCGG AGGATATCTG TATCGGCCAA GTAAACATTT TACCATCATC   500               501   TATCGAGAAG GCCGGTAATT ACTTAGCTGC GCACTACCAA AACTTAAGAC   550               551   GCCCATACTC AGTTGCGATT GCAGGGTATG CGCTCGCGCA CTTGGACAAG   600               601   CTCGAAGGCG ATAATCTGAG GAAATTCTTA AACACTGCCC GTGACCGCAA   650               651   CAGATGGGTC GAGCCAGGAA AAAAGTTGTA CAACGTCGAA GCTACCTCAT   700               701   ATGCATTACT TGCCTTACTC CTTTTGAAAG ATTTTGATAA TGTACCTCCA   750               751   GTAGTTCGTT GGTTGAACGA GCAAAGGTAT TACGGGGGCG GTTATGGTAG   800               801   TACTCAGGCG ACGTTTATGG TATTCCAGGC ATTAGCACAG TACCAAAAGG   850               851   ACGTACCTAA CCACAAAGAC TTAAACCTTC AGGTCTCAAT TAACTTACCT   900          
 
     [0131] SEQID 25 Rhesus macaque (Macaca mulatta) C3d  
                          ACCCCCTCGGGCTGCGGAGAACAGAACATGATCACCATGACGCCCACAGT                   CATCGCTGTGCATTACCTGGATGAAACGGAACAGTGGGAGAAGTTCGGCC               CGGAGAAGCGGCAGGGGGCCTTGGAGCTCATCAAGAAGGGGTACACCCAG               CAGCTGGCCTTCAGACAACCCAGCTCTGCCTTTGCGGCCTTCCTGAACCG               GGCACCCAGCACCTGGCTGACCGCCTACGTGGTCAAGGTCTTCTCTCTGG               CTGTCAACCTCATTGCCATCGACTCCCAGGTCCTCTGCGGGGCTGTTAAA               TGGCTGATCCTGGAGAAGCAGAAGCCCGACGGGGTCTTCCAGGAGGATGC               GCCCGTGATACATCAAGAAATGACTGGTGGATTCCGGAACACCAACGAGA               AAGACATGGCCCTCACGGCCTTTGTTCTCATCTCGCTGCAAGAGGCTAAA               GAGATTTGCGAGGAGCAGGTCAACAGCCTGCCCGGCAGCATCACTAAAGC               AGGAGACTTCCTTGAAGCCAACTACATGAACCTACAGAGATCCTACACTG               TGGCCATCGCTGCCTATGCCCTGGCCCAGATGGGCAGGCTGAAGGGACCT               CTTCTCAACAAATTTCTGACCACAGCCAAAGATAAGAACCGCTGGGAGGA               GCCTGGTCAGCAGCTCTACAATGTGGAGGCCACATCCTATGCCCTCTTGG               CCCTACTGCAGCTAAAAGACTTTGACTTTGTGCCTCCCGTCGTGCGTTGG               CTCAATGAACAGAGATACTACGGTGGTGGCTATGGCTCTACCCAGGCCAC               CTTCATGGTGTTCCAAGCCTTGGCTCAATACCAAAGGATGTCCCTGATCA               CAAGGAACTGAACCTGGATGTGTCCCTCCAACTGCCC          
 
     [0132] SEQID 26 First variant Rhesus C3d  
                          ACGCCAAGCGGATCAGGCGAGCAGAATATGATCACTATGACACCAACCGT                   AATTGCGGTCCATTATCTCGACGAAACCGAACAGTGGGAAAAATTTGGGC               CGGAAAAGCGTCAAGGCGCTCTCGAGTTGATCAAGAAAGGCTACACGCAA               CAGTTAGCGTTCCGTCAACCATCATCAGCGTTCGCCGCTTTCCTGAATCG               TGCGCCATCAACGTGGCTCACAGCGTATGTAGTGAAGGTATTTAGCCTCG               CCGTAAATTTAATCGCGATTGACAGTCAAGTGTTATGCGGCGCGGTCAAG               TGGCTCATTCTTGAAAAGCAAAAGCCAGATGGCGTATTCCAAGAGGACGC               CCCAGTCATCCACCAAGAGATGACAGGCGGCTTTCGCAATACTAATGAGA               AGGACATGGCGTTAACCGCGTTTGTCTTAATCAGTTTACAGGAAGCCAAA               GAAATTTGTGAGGAACAGGTAAATAGTTTACCTGGGAGTATTACGAAAGC               GGGCGATTTCTTAGAAGCAAATTACATGAATCTCCAACGCTCATACACGG               TAGCGATCGCGGCTTATGCCTTAGCGCAGATGGGGAGATTAAAAGGCCCA               TTACTGAACAAGTTTTTAACAACCGCAAAAGACAAGAATAGGTGGGAGGA               ACCAGGCCAACAACTTTATAACGTCGAAGCAACGTCATACGCATTATTAG               CACTCTTACAACTCAAGGACTTCGACTTCGTACCACCTGTGGTACGGTGG               CTTAACGAACAAAGGTATTACGGGGGCGGATACGGCAGCACGCAAGCGAC               TTTCATGGTCTTTCAAGCACTCGCACAGTACCAGAAGGATGTTCCTGATC               ACAAGGAATTAAACTTAGATGTCAGTCTGCAGTTACCA          
 
     [0133] SEQID 27 Second variant Rhesus C3d  
                          ACTCCTTCAGGGAGTGGAGAACAAAACATGATTACAATGACCCCTACAGT                   GATCGCCGTACACTACTTAGATGAGACAGAGCAATGGGAGAAATTCGGTC               CCGAGAAAAGACAGGGAGCGTTAGAACTTATTAAAAAGGGATATACACAG               CAACTCGCTTTTAGGCAGCCTAGTAGCGCATTTGCTGCGTTTCTCAACAG               AGCCCCTAGTACATGGTTAACGGCTTACGTCGTAAAAGTGTTCTCATTAG               CGGTGAACCTGATTGCAATCGATTCGCAGGTACTGTGTGGAGCCGTGAAA               TGGTTAATCTTAGAGAAACAGAAACCTGACGGAGTGTTTCAGGAAGATGC               ACCTGTAATTCACCAGGAAATGACCGGGGGCTTCAGAAACACAAACGAAA               AAGATATGGCTCTGACAGCTTTCGTGCTGATTTCCCTCCAAGAGGCGAAG               GAGATCTGCGAAGAGCAAGTGAACTCACTCCCAGGATCAATCACCAAGGC               CGGGGACTTTCTGGAGGCGAACTATATGAACTTGCAGAGGAGCTATACCG               TCGCAATTGCCGCATACGCGCTCGCACAAATGGGACGTCTCAAAGGACCT               CTGTTAAATAAATTCCTCACGACGGCGAAGGATAAAAACCGATGGGAAGA               ACCTGGGCAACAGTTGTACAATGTAGAGGCGACCAGTTATGCGCTGCTCG               CGTTGCTCCAGTTGAAAGATTTTGATTTTGTCCCTCCAGTAGTCAGATGG               TTGAATGAGCAGCGTTACTATGGAGGCGGGTATGGATCAACACAGGCAAC               GTTTATGGTATTCCAGGCGTTAGCGCAATATCAAAAAGACGTGCCAGACC               ACAAAGAGCTTAATCTCGACGTATCATTACAACTCCCT          
 
     [0134] SEQID 28 Third variant Rhesus C3d  
                          ACACCGTCTGGTAGCGGTGAGCAAAATATGATAACCATGACTCCGACTGT                   TATAGCAGTTCACTATTTAGACGAGACTGAACAATGGGAAAAGTTTGGAC               CGGAAAAAAGGCAAGGTGCACTGGAATTAATAAAAAAAGGTTATACGCAG               CAACTAGCGTTCAGGCAGCCGTCCAGCGCTTTCGCAGCATTTCTGAACAG               GGCTCCGTCCACTTGGTTGACGGCATATGTCGTGAAAGTTTTTAGTTTGG               CAGTTAACTTGATAGCGATCGATAGCCAGGTTTTGTGTGGTGCAGTAAAG               TGGTTGATACTCGAAAAGCAAAAGCCGGATGGTGTTTTTCAAGAAGACGC               CCCGGTTATCCATCAGGAGATGACTGGAGGTTTCAGGAATACCAATGAAA               AGGATATGGCATTGACTGCATTCGTATTGATAAGCTTGCAAGAAGCAAAG               GAGATATGTGAAGAACAAGTTAATTCCTTGCCGGGTTCCATAACAAAGGC               TGGTGATTTTCTCGAGGCTAATTATATGAATCTGCAACGAAGTTATACAG               TTGCTATAGCAGCCTACGCACTCGCTCAAATGGGTCGCTTGAAGGGTCCG               CTCCTGAATAAGTTCTTGACTACTGCTAAGGACAAAAATAGATGGGAAGA               GCCGGGACAGCAACTGTATAATGTTGAAGCTACTAGCTACGCTTTGCTGG               CTCTGTTGCAACTGAAGGATTTCGATTTCGTTCCCCCGGTTGTTAGGTGG               TTAAACGAGCAACGCTATTATGGCGGAGGTTACGGGTCGACTCAAGCTAC               ATTTATGGTTTTTCAGGCTCTGGCCCAGTATCAGAAAGATGTCCCCGATC               ATAAGGAGCTCAATCTGGACGTTAGCTTGCAGTTGCCG          
 
     [0135] SEQID 29 Rhesus C3d amino acid sequence (single letter code)  
                          TPSGSGEQNMITMTPTVIAVHYLDETEQWEKFGPEKRQGALELIKKGYTQ                   QLAFRQPSSAFAAFLNRAPSTWLTAYVVKVFSLAVNLIAIDSQVLCGAVK               WLILEKQKPDGVFQEDAPVIHQEMTGGFRNTNEKDMALTAFVLISLQEAK               EICEEQVNSLPGSITKAGDFLEANYMNLQRSYTVAIAAYALAQMGRLKGP               LLNKFLTTAKDKNRWEEPGQQLYNVEATSYALLALLQLKDFDFVPPVVRW               LNEQRYYGGGYGSTQATFMVFQALAQYQKDVPDHKELNLDVSLQLP          
 
     [0136] SEQID 30 Rhesus C3d amino acid sequence (three letter code)  
                          ThrProSerGlySerGlyGluGlnAsnMetIleThrMetThrProThr                   ValIleAlaVaLHisTyrLeuAspGluThrGluGlnTrpGluLysPhe               GlyProGluLysArgGlnGlyAlaLeuGluLeuIleLysLysGlyTyr               ThrGlnGlnLeuAlaPheArgGlnProSerSerAlaPheAlaAlaPhe               LeuAsnArgAlaProSerThrTrpLeuThrAlaTyrValValLysVal               PheSerLeuAlaValAsnLeuIleAlaIleAspSerGlnValLeuCys               GlyAlaValLysTrpLeuIleLeuGluLysGlnLysproAspGlyVal               PheGlnGluAspAlaProValIleHisGlnGluMetThrGlyGlyPhe               ArgAsnThrAsnGluLysAspMetAlaLeuThrAlaPheValLeuIle               SerLeuGlnGluAlaLysGluIleCysGluGluGlnValAsnSerLeu               ProGlySerIleThrLysAlaGlyAspPheLeuGluAlaAsnTyrMet               AsnLeuGlnArgSerTyrThrValAlaIleAlaAlaTyrAlaLeuAla               GlnMetGlyArgLeuLysGlyProLeuLeuAsnLysPheLeuThrThr               AlaLysAspLysAsnArgTrpGluGluProGlyGlnGlnLeuTyrAsn               ValGluAlaThrSerTyrAlaLeuLeuAlaLeuLeuGlnLeuLysAsp               PheAspPheValProProValValArgTrpLeuAsnGluGlnArgTyr               TyrGlyGlyGlyTyrGlySerThrGlnAlaThrPheMetValPheGln               AlaLeuAlaGlnTyrGlnLysAspValProAspHisLysGluLeuAsn               LeuAspValSerLeuGlnLeuPro          
 
     [0137] 
    
     
       
         1 
         
           
             30  
           
           
             1  
             27  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 1  
             
           
            1 

tgyggrgarc agaacatgat yggcatg                                         27 

 
           
             2  
             26  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 2  
             
           
            2 

ccgtagtatc tyasntcrtt gagcca                                          26 

 
           
             3  
             23  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 3  
             
           
            3 

ggagtcttcg aggagaatgg gcc                                             23 

 
           
             4  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 4  
             
           
            4 

gtgtgtcwgg rrcraagccr gtcatcat                                        28 

 
           
             5  
             27  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 5  
             
           
            5 

gtratgcagg acttcttcat ygacctg                                         27 

 
           
             6  
             24  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 6  
             
           
            6 

ggctgtcagg gacacgtctt tctc                                            24 

 
           
             7  
             25  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 7  
             
           
            7 

gcaagggacc ccmgtggccc agatg                                           25 

 
           
             8  
             23  
             DNA  
             Artificial Sequence  
             
               PCR primer FARM 8  
             
           
            8 

gycaccaccg acaakgtgcc ttg                                             23 

 
           
             9  
             300  
             PRT  
             Bos sp.  
           
            9 

His Leu Ile Gln Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Ser Thr Asp Gln 
            20                  25                  30 

Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Glu Ser Leu Glu Leu Ile 
        35                  40                  45 

Arg Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln Lys Ser Ser Ala 
    50                  55                  60 

Tyr Ala Ala Phe Gln Tyr Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

Val Val Lys Val Phe Ala Leu Ala Ala Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

Lys Asp Leu Cys Glu Thr Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

Pro Asp Gly Ile Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

Ile Gly Gly Phe Arg Asp Thr Arg Glu Lys Asp Val Ser Leu Thr Ala 
    130                 135                 140 

Phe Val Leu Ile Ala Leu His Glu Ala Lys Asp Ile Cys Glu Ala Gln 
145                 150                 155                 160 

Val Asn Ser Leu Gly Arg Ser Ile Ala Lys Ala Gly Asp Phe Leu Glu 
                165                 170                 175 

Asn His Tyr Arg Glu Leu Arg Arg Pro Tyr Thr Val Ala Ile Ala Ala 
            180                 185                 190 

Tyr Ala Leu Ala Leu Leu Gly Lys Leu Glu Gly Asp Arg Leu Thr Lys 
        195                 200                 205 

Phe Leu Asn Thr Ala Lys Glu Lys Asn Arg Trp Glu Glu Pro Asn Gln 
    210                 215                 220 

Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

Ala Arg Lys Asp Tyr Asp Thr Thr Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His 
        275                 280                 285 

Lys Glu Leu Asn Leu Asp Val Ser Ile Gln Leu Pro 
    290                 295                 300 

 
           
             10  
             900  
             DNA  
             Bos sp.  
             
               CDS  
               (1)..(900)  
             
           
            10 

cac ctt atc caa acc ccc tcc ggc tgt ggg gag cag aac atg att ggt       48 
His Leu Ile Gln Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

atg acg ccc acg gtc atc gcc gtg cac tac ctg gac agc acc gac cag       96 
Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Ser Thr Asp Gln 
            20                  25                  30 

tgg gag aag ttc ggc ttg gag aag cgg cag gag tcc ctg gag ctc atc      144 
Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Glu Ser Leu Glu Leu Ile 
        35                  40                  45 

aga aag ggg tac acc cag cag ctg gcc ttc aga caa aaa agc tca gcc      192 
Arg Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln Lys Ser Ser Ala 
    50                  55                  60 

tac gcc gcc ttc caa tat cgg ccc ccc agc acc tgg ctg aca gcc tac      240 
Tyr Ala Ala Phe Gln Tyr Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

gtg gtc aag gtc ttt gca ctg gcc gcc aac ctc atc gcc ata gac tcc      288 
Val Val Lys Val Phe Ala Leu Ala Ala Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

aag gac ctc tgt gag acc gtc aaa tgg ctg atc ctg gag aag cag aag      336 
Lys Asp Leu Cys Glu Thr Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

cct gat gga atc ttc cag gag gat ggg cct gtg ata cac caa gaa atg      384 
Pro Asp Gly Ile Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

att ggt ggc ttc agg gac acc agg gag aaa gat gtg tcc ctt aca gcc      432 
Ile Gly Gly Phe Arg Asp Thr Arg Glu Lys Asp Val Ser Leu Thr Ala 
    130                 135                 140 

ttt gtt ctc atc gcg ctg cac gag gct aaa gac att tgc gag gca cag      480 
Phe Val Leu Ile Ala Leu His Glu Ala Lys Asp Ile Cys Glu Ala Gln 
145                 150                 155                 160 

gtc aac agc ctg ggc cgc agc atc gct aag gca gga gac ttc ctc gaa      528 
Val Asn Ser Leu Gly Arg Ser Ile Ala Lys Ala Gly Asp Phe Leu Glu 
                165                 170                 175 

aac cac tac aga gag ttg cga aga cca tat act gtg gcc att gct gcc      576 
Asn His Tyr Arg Glu Leu Arg Arg Pro Tyr Thr Val Ala Ile Ala Ala 
            180                 185                 190 

tat gcc ctg gct ttg ttg ggc aag ctg gag ggt gac cgc ctc acc aaa      624 
Tyr Ala Leu Ala Leu Leu Gly Lys Leu Glu Gly Asp Arg Leu Thr Lys 
        195                 200                 205 

ttt ctg aac aca gcc aaa gaa aag aac cgc tgg gag gaa ccc aac cag      672 
Phe Leu Asn Thr Ala Lys Glu Lys Asn Arg Trp Glu Glu Pro Asn Gln 
    210                 215                 220 

aag ctc tac aat gtg gag gcc acg tcc tac gcc ctc ttg gct ctg ctg      720 
Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

gca cgc aaa gac tac gac act acg cct cct gtc gtg cgc tgg ctc aac      768 
Ala Arg Lys Asp Tyr Asp Thr Thr Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

gag cag aga tac tat gga ggt ggt tat ggc tcc acg cag gcc act ttc      816 
Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

atg gtg ttc caa gcc ttg gcc caa tac cag aag gat gtt cct gat cac      864 
Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His 
        275                 280                 285 

aag gag ctg aac ctg gat gtg tcc atc caa ctg ccc                      900 
Lys Glu Leu Asn Leu Asp Val Ser Ile Gln Leu Pro 
    290                 295                 300 

 
           
             11  
             300  
             PRT  
             Equus caballus  
           
            11 

His Leu Ile Val Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Ser 
1               5                   10                  15 

Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Gln Thr Glu Gln 
            20                  25                  30 

Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Glu Ser Leu Glu Leu Ile 
        35                  40                  45 

Lys Lys Gly Tyr Thr Gln Gln Leu Ala Tyr Arg Gln Pro Ser Ser Ala 
    50                  55                  60 

Tyr Ala Ala Phe Leu Ser Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

Val Val Lys Val Phe Ala Leu Ala Ser Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Gln Lys Gln Lys 
            100                 105                 110 

Pro Asp Gly Val Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

Ile Gly Gly Phe Arg Asn Ala Glu Glu Lys Asp Val Ser Leu Thr Ala 
    130                 135                 140 

Phe Val Leu Ile Ala Leu Gln Glu Ala Lys Asp Ile Cys Glu Gly Gln 
145                 150                 155                 160 

Val Asn Ser Leu Ala Arg Ser Ile Ile Lys Ala Gly Asp Phe Leu Glu 
                165                 170                 175 

Ala His Tyr Asn Asn Leu Arg Arg Pro Tyr Ser Val Ala Ile Ala Gly 
            180                 185                 190 

Tyr Ala Leu Ala Gln Met Gly Lys Leu Glu Asp Pro Leu Leu Asn Lys 
        195                 200                 205 

Phe Leu Ser Ala Ala Thr Asp Arg Asn Arg Trp Glu Glu Pro Gly Gln 
    210                 215                 220 

Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

Leu Leu Arg Asp Phe Asp Ser Val Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asn His 
        275                 280                 285 

Lys Asp Leu Asn Leu Asp Val Ser Ile Asn Leu Pro 
    290                 295                 300 

 
           
             12  
             900  
             DNA  
             Equus caballus  
             
               CDS  
               (1)..(900)  
             
           
            12 

cac ctc atc gtg acg ccc tcg ggc tgc ggc gag cag aac atg att agc       48 
His Leu Ile Val Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Ser 
1               5                   10                  15 

atg acg ccc acg gtc atc gca gtg cat tac ctg gac cag acc gag cag       96 
Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Gln Thr Glu Gln 
            20                  25                  30 

tgg gag aag ttc ggc ctg gag aag cgg cag gag tcc ttg gag ctc atc      144 
Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Glu Ser Leu Glu Leu Ile 
        35                  40                  45 

aag aag ggg tac acc cag cag ctg gcc tac aga caa ccc agc tca gcc      192 
Lys Lys Gly Tyr Thr Gln Gln Leu Ala Tyr Arg Gln Pro Ser Ser Ala 
    50                  55                  60 

tat gca gcc ttc ctg agc cgg ccg ccc agc acc tgg ctg aca gcc tac      240 
Tyr Ala Ala Phe Leu Ser Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

gtg gtc aag gtc ttc gct ctg gcc tcc aac ctc atc gcc atc gac tcc      288 
Val Val Lys Val Phe Ala Leu Ala Ser Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

cag gtc ctc tgt ggg gct gtc aaa tgg ctg atc ctc cag aag cag aag      336 
Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Gln Lys Gln Lys 
            100                 105                 110 

cca gat gga gtc ttc cag gag gac ggg ccc gtg ata cat caa gaa atg      384 
Pro Asp Gly Val Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

att ggt ggc ttc cgg aat gcg gag gag aaa gac gtg tcc ctc aca gcc      432 
Ile Gly Gly Phe Arg Asn Ala Glu Glu Lys Asp Val Ser Leu Thr Ala 
    130                 135                 140 

ttt gtt ctc atc gca ctg cag gaa gct aaa gat att tgc gag gga cag      480 
Phe Val Leu Ile Ala Leu Gln Glu Ala Lys Asp Ile Cys Glu Gly Gln 
145                 150                 155                 160 

gtc aac agc ctg gca cgc agc atc att aag gca gga gac ttc ctt gaa      528 
Val Asn Ser Leu Ala Arg Ser Ile Ile Lys Ala Gly Asp Phe Leu Glu 
                165                 170                 175 

gcc cac tat aat aac ctg cgg aga cca tat tct gtg gcc att gct ggc      576 
Ala His Tyr Asn Asn Leu Arg Arg Pro Tyr Ser Val Ala Ile Ala Gly 
            180                 185                 190 

tac gcc ctg gcc cag atg ggc aag ctg gag gac ccc ctc ctc aac aaa      624 
Tyr Ala Leu Ala Gln Met Gly Lys Leu Glu Asp Pro Leu Leu Asn Lys 
        195                 200                 205 

ttc ctg agc gca gcc aca gac agg aac cgc tgg gag gag cct ggc cag      672 
Phe Leu Ser Ala Ala Thr Asp Arg Asn Arg Trp Glu Glu Pro Gly Gln 
    210                 215                 220 

aag ctc tac aat gta gag gcc aca tcc tac gcc ctc ttg gcc ctg ctg      720 
Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

ctg ctc aga gac ttt gac tct gtg cct ccg gtg gtg cgc tgg ctc aac      768 
Leu Leu Arg Asp Phe Asp Ser Val Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

gaa cag aga tac tac gga ggt ggc tat ggc tcc acc cag gcc acc ttc      816 
Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

atg gtg ttc caa gcc ttg gct cag tac caa aag gat gtc cct aac cac      864 
Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asn His 
        275                 280                 285 

aag gac ctg aac ctc gat gtt tcc atc aac ctg ccc                      900 
Lys Asp Leu Asn Leu Asp Val Ser Ile Asn Leu Pro 
    290                 295                 300 

 
           
             13  
             300  
             PRT  
             Callithrix sp.  
           
            13 

His Leu Ile Val Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Gln Thr Glu Gln 
            20                  25                  30 

Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Gly Ala Val Glu Leu Ile 
        35                  40                  45 

Lys Lys Gly Tyr Ala Gln Gln Leu Ala Phe Lys Gln Pro Ser Ser Ala 
    50                  55                  60 

Tyr Ala Ala Phe Leu Asn Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

Val Val Lys Val Phe Ser Leu Ala Val Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

Pro Asp Gly Val Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

Ile Gly Gly Phe Arg Asn Thr Gln Glu Lys Asp Met Ala Leu Thr Ala 
    130                 135                 140 

Phe Val Leu Ile Ser Leu Gln Glu Ala Lys Asp Ile Cys Glu Glu Leu 
145                 150                 155                 160 

Val Asn Ser Leu Pro Arg Ser Ile Ile Asp Ala Gly Asn Phe Leu Glu 
                165                 170                 175 

Ala Asn Tyr Met Asn Leu Gln Arg Ser Tyr Thr Val Ala Ile Ala Gly 
            180                 185                 190 

Tyr Ala Leu Ala Gln Leu Asp Lys Leu Asn Gly Pro Leu Leu Asn Lys 
        195                 200                 205 

Phe Leu Ser Thr Ala Lys Asp Lys Asn Arg Trp Glu Glu Pro Gly Gln 
    210                 215                 220 

Gln Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

Gln Met Lys Asp Phe Asp Phe Val Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His 
        275                 280                 285 

Lys Glu Leu Asn Leu Asp Val Ser Leu Gln Leu Pro 
    290                 295                 300 

 
           
             14  
             900  
             DNA  
             Callithrix sp.  
             
               CDS  
               (1)..(900)  
             
           
            14 

cac ctc atc gtg acc ccc tcg ggc tgt ggg gaa cag aac atg att ggc       48 
His Leu Ile Val Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

atg aca ccc acg gtc atc gcg gtg cat tac ctg gat caa acg gag cag       96 
Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Gln Thr Glu Gln 
            20                  25                  30 

tgg gag aag ttc ggc ttg gag aag cgg cag gga gcc gtg gag ctc atc      144 
Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Gly Ala Val Glu Leu Ile 
        35                  40                  45 

aag aag ggg tac gca cag cag ctg gcc ttc aaa caa ccc agc tct gcc      192 
Lys Lys Gly Tyr Ala Gln Gln Leu Ala Phe Lys Gln Pro Ser Ser Ala 
    50                  55                  60 

tat gcg gcc ttc ctg aac cgg cca ccc agc acc tgg ctg acc gcc tac      240 
Tyr Ala Ala Phe Leu Asn Arg Pro Pro Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

gtg gtc aag gtc ttc tct ctg gcc gtc aac ctc att gcc att gac tcc      288 
Val Val Lys Val Phe Ser Leu Ala Val Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

caa gtc ctc tgt ggg gct gtt aaa tgg ctg atc ctg gag aag cag aag      336 
Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

cct gat ggg gtc ttc cag gag gat ggg ccc gtg ata cac caa gaa atg      384 
Pro Asp Gly Val Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

att ggt ggc ttc cgg aac acc cag gag aaa gac atg gcc ctc acg gcc      432 
Ile Gly Gly Phe Arg Asn Thr Gln Glu Lys Asp Met Ala Leu Thr Ala 
    130                 135                 140 

ttt gtt ctc atc tcg ctg cag gag gct aaa gat att tgc gag gag ctt      480 
Phe Val Leu Ile Ser Leu Gln Glu Ala Lys Asp Ile Cys Glu Glu Leu 
145                 150                 155                 160 

gtc aac agc ctg cca cgc agc atc att gat gca gga aac ttc ctt gaa      528 
Val Asn Ser Leu Pro Arg Ser Ile Ile Asp Ala Gly Asn Phe Leu Glu 
                165                 170                 175 

gcc aac tac atg aac cta cag aga tcc tac act gtg gcc atc gct ggc      576 
Ala Asn Tyr Met Asn Leu Gln Arg Ser Tyr Thr Val Ala Ile Ala Gly 
            180                 185                 190 

tat gcc ctg gcc cag ctg gac aaa ctg aac ggg cct ctt ctc aac aaa      624 
Tyr Ala Leu Ala Gln Leu Asp Lys Leu Asn Gly Pro Leu Leu Asn Lys 
        195                 200                 205 

ttt ctg agc aca gcc aaa gat aag aac cgc tgg gag gag cct ggt cag      672 
Phe Leu Ser Thr Ala Lys Asp Lys Asn Arg Trp Glu Glu Pro Gly Gln 
    210                 215                 220 

cag ctc tac aac gtg gag gcc aca tcc tat gcc ctc ttg gcc ctc ctg      720 
Gln Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

cag atg aaa gac ttc gac ttt gtg cct ccc gtc gtg cgt tgg ctc aat      768 
Gln Met Lys Asp Phe Asp Phe Val Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

gag cag aga tac tac ggt ggc ggc tat ggc tct acc cag gcc acc ttc      816 
Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

atg gtg ttc caa gcc ttg gcc caa tac caa aag gac gtc cct gac cac      864 
Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His 
        275                 280                 285 

aag gaa ctg aac ctg gat gtg tcc ctc caa ctg cca                      900 
Lys Glu Leu Asn Leu Asp Val Ser Leu Gln Leu Pro 
    290                 295                 300 

 
           
             15  
             300  
             PRT  
             Sus sp.  
           
            15 

His Leu Ile Gln Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Ser Thr Glu Gln 
            20                  25                  30 

Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Glu Ala Leu Glu Leu Ile 
        35                  40                  45 

Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln Lys Asn Ser Ala 
    50                  55                  60 

Phe Ala Ala Phe Gln Asp Arg Leu Ser Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

Val Val Lys Val Phe Ala Met Ala Ala Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

Pro Asp Gly Val Phe Glu Glu Asn Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

Ile Gly Gly Phe Lys Asn Thr Glu Glu Lys Asp Val Ser Leu Thr Ala 
    130                 135                 140 

Phe Val Leu Ile Ala Leu Gln Glu Ala Lys Asp Ile Cys Glu Pro Gln 
145                 150                 155                 160 

Val Asn Ser Leu Leu Arg Ser Ile Asn Lys Ala Arg Asp Phe Leu Ala 
                165                 170                 175 

Asp Tyr Tyr Leu Glu Leu Lys Arg Pro Tyr Thr Val Ala Ile Ala Gly 
            180                 185                 190 

Tyr Ala Leu Ala Leu Ser Asp Lys Leu Asp Glu Pro Phe Leu Asn Lys 
        195                 200                 205 

Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln 
    210                 215                 220 

Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

Val Val Lys Asp Phe Asp Ser Val Pro Pro Ile Val Arg Trp Leu Asn 
                245                 250                 255 

Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His 
        275                 280                 285 

Lys Asp Leu Asn Leu Asp Val Ser Ile His Leu Pro 
    290                 295                 300 

 
           
             16  
             900  
             DNA  
             Sus sp.  
             
               CDS  
               (1)..(900)  
             
           
            16 

cac ctc atc caa acc ccc tcc ggc tgt ggg gag cag aac atg atc ggc       48 
His Leu Ile Gln Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

atg acg ccc aca gtc atc gct gtg cac tac ctg gac agc acc gaa caa       96 
Met Thr Pro Thr Val Ile Ala Val His Tyr Leu Asp Ser Thr Glu Gln 
            20                  25                  30 

tgg gag aag ttc ggc ctg gag aag agg cag gaa gcc ttg gag ctc atc      144 
Trp Glu Lys Phe Gly Leu Glu Lys Arg Gln Glu Ala Leu Glu Leu Ile 
        35                  40                  45 

aag aag ggg tac acc cag caa ctg gcc ttc aga caa aag aac tca gcc      192 
Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln Lys Asn Ser Ala 
    50                  55                  60 

ttt gcc gcc ttc cag gac cgg ctg tcc agc acc tgg ctg aca gcc tat      240 
Phe Ala Ala Phe Gln Asp Arg Leu Ser Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

gtg gtc aag gtc ttc gct atg gca gcc aac ctc atc gcc atc gac tcc      288 
Val Val Lys Val Phe Ala Met Ala Ala Asn Leu Ile Ala Ile Asp Ser 
                85                  90                  95 

cag gtc ctc tgt ggg gcc gtc aaa tgg ctg atc ctg gag aag cag aag      336 
Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

cct gat gga gtc ttc gag gag aat ggg ccc gtg ata cac caa gaa atg      384 
Pro Asp Gly Val Phe Glu Glu Asn Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

att ggt ggc ttc aag aac act gag gag aaa gac gtg tcc ctg aca gcc      432 
Ile Gly Gly Phe Lys Asn Thr Glu Glu Lys Asp Val Ser Leu Thr Ala 
    130                 135                 140 

ttt gtt ctc atc gcg ctg cag gag gct aaa gac atc tgt gaa cca cag      480 
Phe Val Leu Ile Ala Leu Gln Glu Ala Lys Asp Ile Cys Glu Pro Gln 
145                 150                 155                 160 

gtc aat agc ctg ttg cgc agc atc aat aag gca aga gac ttc ctc gca      528 
Val Asn Ser Leu Leu Arg Ser Ile Asn Lys Ala Arg Asp Phe Leu Ala 
                165                 170                 175 

gac tac tac cta gaa tta aaa aga cca tat act gtg gcc att gct ggt      576 
Asp Tyr Tyr Leu Glu Leu Lys Arg Pro Tyr Thr Val Ala Ile Ala Gly 
            180                 185                 190 

tat gcc ctg gct cta tct gac aag ctg gat gag ccc ttc ctc aac aaa      624 
Tyr Ala Leu Ala Leu Ser Asp Lys Leu Asp Glu Pro Phe Leu Asn Lys 
        195                 200                 205 

ctt ctg agc aca gcc aaa gaa agg aac cgc tgg gag gaa cct ggc cag      672 
Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu Glu Pro Gly Gln 
    210                 215                 220 

aag ctc tac aat gtg gag gcc aca tcc tac gcc ctc ttg gct ctg ctg      720 
Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

gta gtc aaa gac ttt gac tct gtc cct cct att gtg cgc tgg ctc aat      768 
Val Val Lys Asp Phe Asp Ser Val Pro Pro Ile Val Arg Trp Leu Asn 
                245                 250                 255 

gag cag aga tac tac gga ggt ggc tac gga tct acc cag gcc act ttc      816 
Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

atg gtg ttc caa gcc ttg gcc caa tac cag aag gat gtc cct gat cac      864 
Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His 
        275                 280                 285 

aag gat ctg aac ctg gat gtg tcc atc cac ctg ccc                      900 
Lys Asp Leu Asn Leu Asp Val Ser Ile His Leu Pro 
    290                 295                 300 

 
           
             17  
             312  
             PRT  
             Canis familiaris  
           
            17 

His Leu Ile Val Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

Met Thr Pro Thr Val Ile Ala Leu His Tyr Leu Asp Gln Thr Glu Gln 
            20                  25                  30 

Trp Asp Lys Phe Gly Leu Glu Lys Arg Gln Glu Ala Leu Glu Leu Ile 
        35                  40                  45 

Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln Pro Asn Ser Ala 
    50                  55                  60 

Phe Ala Ala Phe Gln Asn Arg Pro Ser Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

Val Val Lys Val Phe Ser Leu Ala Thr Asn Leu Ile Ala Ile Glu Ala 
                85                  90                  95 

Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

Pro Asp Gly Ile Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

Thr Gly Gly Phe Arg Glu Ala Glu Glu Lys Ser Val Ser Leu Thr Ala 
    130                 135                 140 

Phe Val Leu Ile Ala Leu Lys Glu Ala Glu Asp Ile Cys Ile Gly Gln 
145                 150                 155                 160 

Val Asn Ile Leu Pro Ser Ser Ile Glu Lys Ala Gly Asn Tyr Leu Ala 
                165                 170                 175 

Ala His Tyr Gln Asn Leu Arg Arg Pro Tyr Ser Val Ala Ile Ala Gly 
            180                 185                 190 

Tyr Ala Leu Ala His Leu Asp Lys Leu Glu Gly Asp Asn Leu Arg Lys 
        195                 200                 205 

Phe Leu Asn Thr Ala Arg Asp Arg Asn Arg Trp Val Glu Pro Gly Lys 
    210                 215                 220 

Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

Leu Leu Lys Asp Phe Asp Asn Val Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asn His 
        275                 280                 285 

Lys Asp Leu Asn Leu Gln Val Ser Ile Asn Leu Pro Ser Arg Ser Thr 
    290                 295                 300 

Glu Ile Lys His His Ile Val Trp 
305                 310 

 
           
             18  
             936  
             DNA  
             Canis familiaris  
             
               CDS  
               (1)..(936)  
             
           
            18 

cac ctc atc gtg acc ccg tcg ggc tgc ggg gag cag aac atg atc ggc       48 
His Leu Ile Val Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly 
1               5                   10                  15 

atg acg ccc acc gtc atc gcc ctg cat tac ctg gac caa acc gag cag       96 
Met Thr Pro Thr Val Ile Ala Leu His Tyr Leu Asp Gln Thr Glu Gln 
            20                  25                  30 

tgg gac aag ttc ggg ctg gag aag cgc cag gag gcc ttg gag ctc atc      144 
Trp Asp Lys Phe Gly Leu Glu Lys Arg Gln Glu Ala Leu Glu Leu Ile 
        35                  40                  45 

aag aag gga tac acc caa cag ctg gcc ttc aga caa ccc aac tcg gcc      192 
Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln Pro Asn Ser Ala 
    50                  55                  60 

ttc gct gcc ttc cag aac cgg cca tcc agc acc tgg ctg aca gcc tac      240 
Phe Ala Ala Phe Gln Asn Arg Pro Ser Ser Thr Trp Leu Thr Ala Tyr 
65                  70                  75                  80 

gtg gtc aag gtc ttc tct ctg gcc acc aac ctc atc gcc att gaa gcc      288 
Val Val Lys Val Phe Ser Leu Ala Thr Asn Leu Ile Ala Ile Glu Ala 
                85                  90                  95 

cag gtt ctc tgc ggg gct gtc aaa tgg ctg atc ctg gag aag cag aag      336 
Gln Val Leu Cys Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys 
            100                 105                 110 

ccc gat ggg atc ttc cag gag gat ggg cct gtg atc cac caa gag atg      384 
Pro Asp Gly Ile Phe Gln Glu Asp Gly Pro Val Ile His Gln Glu Met 
        115                 120                 125 

acc ggt ggc ttc cgg gaa gct gag gag aag tct gtg tcc ctc acg gcc      432 
Thr Gly Gly Phe Arg Glu Ala Glu Glu Lys Ser Val Ser Leu Thr Ala 
    130                 135                 140 

ttt gtt ctc ata gca cta aaa gag gct gaa gat att tgt ata gga cag      480 
Phe Val Leu Ile Ala Leu Lys Glu Ala Glu Asp Ile Cys Ile Gly Gln 
145                 150                 155                 160 

gtt aac atc ttg cca agc agc atc gaa aag gca gga aat tat ctt gca      528 
Val Asn Ile Leu Pro Ser Ser Ile Glu Lys Ala Gly Asn Tyr Leu Ala 
                165                 170                 175 

gcc cac tac cag aac ctg agg aga cca tat tct gtg gcc att gct ggc      576 
Ala His Tyr Gln Asn Leu Arg Arg Pro Tyr Ser Val Ala Ile Ala Gly 
            180                 185                 190 

tac gcc ctg gcc cat cta gac aaa ctg gag gga gat aac ctc aga aaa      624 
Tyr Ala Leu Ala His Leu Asp Lys Leu Glu Gly Asp Asn Leu Arg Lys 
        195                 200                 205 

ttt ctg aac aca gcc aga gac agg aac cgc tgg gtg gag cct ggc aag      672 
Phe Leu Asn Thr Ala Arg Asp Arg Asn Arg Trp Val Glu Pro Gly Lys 
    210                 215                 220 

aag ctc tac aat gtg gaa gcc aca tcc tac gcc ctc ttg gcc ctg ctg      720 
Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu 
225                 230                 235                 240 

ctg ctc aaa gac ttt gac aat gta cct cct gtc gtc cgc tgg ctc aat      768 
Leu Leu Lys Asp Phe Asp Asn Val Pro Pro Val Val Arg Trp Leu Asn 
                245                 250                 255 

gag cag aga tac tac gga ggt ggc tat ggc tcc acc cag gcc acc ttc      816 
Glu Gln Arg Tyr Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe 
            260                 265                 270 

atg gtg ttc caa gcc ttg gcc caa tac cag aag gat gtc ccc aac cac      864 
Met Val Phe Gln Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asn His 
        275                 280                 285 

aag gac ctg aac ctg caa gtg tcc atc aac ctg ccc agc cgc agc aca      912 
Lys Asp Leu Asn Leu Gln Val Ser Ile Asn Leu Pro Ser Arg Ser Thr 
    290                 295                 300 

gag atc aag cac cac att gtc tgg                                      936 
Glu Ile Lys His His Ile Val Trp 
305                 310 

 
           
             19  
             900  
             DNA  
             Artificial Sequence  
             
               Nucleotide sequence of first variant Bos sp. 
      C3d  
             
           
            19 

cacctgattc aaactcctag cgggtgcggc gagcaaaaca tgattgggat gacccctaca     60 

gttatcgcag tccactacct cgattccact gaccaatggg aaaaattcgg actggaaaaa    120 

cgccaagaga gcctcgagtt gattaggaaa ggctacactc agcaactcgc attccgtcag    180 

aaatcttccg cttacgcggc tttccagtat aggcctcctt ccacatggct cactgcatac    240 

gtcgtgaaag tgtttgcctt ggctgctaac ttgatcgcta ttgacagcaa agacctgtgt    300 

gaaacggtga agtggctcat tctcgaaaag caaaagccag atggtatttt tcaagaggac    360 

ggcccagtca ttcaccagga aatgatcggc ggttttcgcg acactagaga gaaggatgtc    420 

agtctcaccg cttttgtgct gattgccctc cacgaagcaa aagatatctg cgaagctcag    480 

gtgaattctc tcgggcgtag tatcgccaag gctggtgatt tcttggagaa ccactaccgt    540 

gagctccgca ggccatatac cgtcgctatt gccgcttatg cactcgccct cctggggaag    600 

ttagagggag atcgtctgac taaattcctc aacacggcaa aagagaagaa tcgctgggaa    660 

gaacctaacc aaaagttgta taatgttgag gcaaccagct acgcactgct ggcactcctc    720 

gctaggaagg actatgacac cactcctcca gtggtccgtt ggttgaacga acagcgctac    780 

tacggtggcg gctatgggtc tacccaggct acatttatgg tcttccaggc tctggctcaa    840 

tatcagaaag atgtcccaga tcacaaggaa ctcaatctcg atgtttctat ccaattgcct    900 

 
           
             20  
             900  
             DNA  
             Artificial Sequence  
             
               Nucleotide sequence of second variant Bos sp. 
      C3d  
             
           
            20 

cacttgatcc agacaccatc tggttgtgga gaacaaaata tgatcggcat gacaccaacc     60 

gtgattgctg ttcactattt ggatagtaca gatcagtggg aaaagtttgg gctcgagaaa    120 

agacaggaat ctcttgaact gatccgcaaa ggatatacac aacagttggc ttttcgccaa    180 

aagtccagcg catatgcagc atttcaatac cgcccaccat ctacttggtt gaccgcttat    240 

gttgttaagg ttttcgctct cgcagcaaat ctgattgcaa tcgattctaa ggatttgtgc    300 

gagactgtta aatggttaat cttggagaaa cagaaacctg acgggatctt tcaggaagat    360 

ggtcctgtta tccaccagga gatgatcggg ggatttagag atacacgtga aaaagacgtt    420 

tctctgactg cattcgtctt gatcgctttg cacgaggcca aggacatctg tgaggcccaa    480 

gttaatagtt tgggtagatc cattgcaaaa gccggggact ttctggaaaa tcactatagg    540 

gaactgagac gcccttacac agtagcaatc gcagcatacg ctttggcact gctcggaaaa    600 

ctcgaagggg acagattaac aaagtttttg aataccgcta aggaaaaaaa cagatgggaa    660 

gagccaaatc agaaactgta caacgtagaa gctactagtt atgctttgct cgccttgttg    720 

gccagaaaag attacgatac aaccccacct gtagtaagat ggctgaatga gcaaaggtat    780 

tacgggggag gatacggaag cactcaagca accttcatgg tttttcaagc actcgcacag    840 

taccaaaagg acgttcctga ccacaaagag ttgaacttgg acgtcagcat tcagctccca    900 

 
           
             21  
             900  
             DNA  
             Artificial Sequence  
             
               Nucleotide sequence of first variant Sus sp. 
      C3d  
             
           
            21 

cacctgattc aaacgccatc agggtgcgga gagcaaaaca tgattggtat gaccccaacg     60 

gtgatcgcag tccactatct cgattcaacg gaacagtggg agaaattcgg attagagaaa    120 

agacaagaag cgctcgaatt gatcaaaaag ggctatacgc agcagttagc ttttagacag    180 

aagaattccg cgttcgctgc gttccaagac agacttagtt caacatggtt aactgcgtat    240 

gttgtgaaag ttttcgccat ggctgcgaat ctgattgcga tcgattcaca agtgttatgt    300 

ggcgctgtga aatggttaat tcttgaaaag caaaagccag atggcgtgtt tgaggaaaat    360 

ggcccagtca ttcaccagga aatgatcggc ggatttaaga atacggagga aaaagatgta    420 

tcattaacgg catttgtctt aattgcactt caagaggcga aggacatttg tgagcctcag    480 

gtgaacagtt tattaagaag tattaacaag gcgcgtgact ttctggcgga ttactatctc    540 

gagttgaaga ggccttatac ggtagctatc gcgggatacg cactcgcctt gtcagataag    600 

ttagatgaac catttctgaa taaattactt tcaacggcaa aagagagaaa tcgttgggaa    660 

gaacctggac aaaagttata taacgtagag gcaacgagtt acgcacttct tgccctctta    720 

gtggttaagg acttcgacag cgtacctcca attgtcagat ggctgaacga gcaacgctac    780 

tatgggggag ggtacggcag cacgcaggca acgtttatgg tcttccaggc gttagcgcag    840 

taccaaaagg acgtaccaga tcacaaagat ttaaacctcg acgtcagtat ccacttacca    900 

 
           
             22  
             900  
             DNA  
             Artificial Sequence  
             
               Nucleotide sequence of second variant Sus sp. 
      C3d  
             
           
            22 

cacttaatcc agactcctag tggatgtggc gaacagaata tgatcgggat gacacctacc     60 

gtaattgcgg ttcactactt agacagtaca gagcaatggg aaaagtttgg gctcgaaaag    120 

cgccaggagg ctcttgagtt aattaagaaa ggttacacac aacaactcgc attcaggcaa    180 

aaaaacagtg catttgcggc atttcaggat cgcttaagca gtacgtggct caccgcatac    240 

gtcgtaaagg tgtttgcgat ggccgcaaac ttaatcgcaa ttgacagtca ggtactgtgc    300 

ggagcggtta agtggcttat cttagagaaa cagaaacctg acggggtatt cgaagagaac    360 

ggtcctgtta tccaccaaga gatgattggg ggtttcaaaa acaccgaaga gaaggacgtt    420 

agtctcaccg ctttcgtgct gatcgcctta caggaagcca aagatatctg cgaaccacaa    480 

gtaaattcac tccttcgttc aatcaataaa gccagggatt tcttagccga ctattacttg    540 

gaactcaaac gtccatacac cgttgcgatt gccgggtatg ctttagcgct cagcgacaaa    600 

ctcgacgagc ctttcttaaa caagctgtta agtaccgcta aggaacgcaa caggtgggag    660 

gagccaggtc agaaacttta caatgttgaa gctacctcat atgctttatt agcgttactc    720 

gtcgtaaaag attttgattc agtgccacct atcgtacgtt ggttaaatga acagaggtat    780 

tacggtggcg gatatgggtc aacacaagcg accttcatgg tatttcaagc actcgcacaa    840 

tatcagaaag atgtgcctga ccacaaggac ctgaatttag atgtatcaat tcaccttcct    900 

 
           
             23  
             900  
             DNA  
             Artificial Sequence  
             
               Nucleotide sequence of first variant Canis 
      familiaris C3d  
             
           
            23 

cacttaatcg tcactccaag tggatgcggc gaacagaata tgatcggaat gacaccaacg     60 

gtaattgcgc tccactattt agaccagact gagcaatggg ataagtttgg actcgaaaag    120 

aggcaggaag cgctcgagtt aatcaaaaag ggctatacgc agcagttagc gtttaggcag    180 

ccaaattccg cgtttgcagc gttccaaaac agaccatcat caacgtggct tacggcttat    240 

gtcgttaaag tgttctcatt agcgactaat cttattgcaa tcgaggctca ggtcctttgt    300 

ggcgcggtta aatggttaat tctcgaaaag caaaaaccag acggcatttt ccaagaggac    360 

ggccctgtaa ttcaccaaga aatgacgggc ggttttcgcg aagcggaaga gaaatcagta    420 

agtcttaccg cgttcgtgct gattgcgttg aaagaagccg aagacatttg cattgggcag    480 

gtcaatatcc tccctagttc aattgaaaaa gctggcaact atctggccgc acactatcag    540 

aatctccgta ggccttatag cgtcgcaata gccggttacg cattagcaca cctcgataaa    600 

ttagagggtg acaacttacg caagtttctc aatacggcaa gggatagaaa tcgttgggta    660 

gaaccaggga agaaacttta taatgtagag gcgacaagtt acgcgctgct cgctctttta    720 

ttactaaagg acttcgacaa cgtcccacct gtggtgaggt ggcttaatga acagcgttac    780 

tatggcggag gatacggatc aacgcaagct acattcatgg tctttcaagc gctcgcgcaa    840 

tatcagaaag atgtgccaaa tcacaaggat ctcaatttac aagtaagtat caatctccca    900 

 
           
             24  
             900  
             DNA  
             Artificial Sequence  
             
               Nucleotide sequence of second variant Canis 
      familiaris C3d  
             
           
            24 

cacctgattg taacgcctag cggttgtgga gagcaaaaca tgattgggat gacccctact     60 

gtgatcgcat tacactacct cgatcaaaca gaacagtggg acaaattcgg tttagagaaa    120 

cgtcaagagg ctttagaact gattaagaaa ggttacaccc aacaactcgc tttccgtcaa    180 

cctaacagtg ctttcgcggc ttttcagaat cgtcctagta gtacatggtt aaccgcatac    240 

gtagtgaagg tatttagtct tgcaacgaac ttaatcgcta tcgaagcgca agtgttgtgc    300 

ggagccgtga agtggctcat cttagagaaa cagaagcctg atggtatctt tcaggaagat    360 

ggaccagtca tccaccagga gatgactgga gggttcagag aggccgagga aaagagcgtg    420 

tcattgacag cttttgtctt aatcgccctc aaggaggcgg aggatatctg tatcggccaa    480 

gtaaacattt taccatcatc tatcgagaag gccggtaatt acttagctgc gcactaccaa    540 

aacttaagac gcccatactc agttgcgatt gcagggtatg cgctcgcgca cttggacaag    600 

ctcgaaggcg ataatctgag gaaattctta aacactgccc gtgaccgcaa cagatgggtc    660 

gagccaggaa aaaagttgta caacgtcgaa gctacctcat atgcattact tgccttactc    720 

cttttgaaag attttgataa tgtacctcca gtagttcgtt ggttgaacga gcaaaggtat    780 

tacgggggcg gttatggtag tactcaggcg acgtttatgg tattccaggc attagcacag    840 

taccaaaagg acgtacctaa ccacaaagac ttaaaccttc aggtctcaat taacttacct    900 

 
           
             25  
             888  
             DNA  
             Macaca mulatta  
           
            25 

accccctcgg gctgcggaga acagaacatg atcaccatga cgcccacagt catcgctgtg     60 

cattacctgg atgaaacgga acagtgggag aagttcggcc cggagaagcg gcagggggcc    120 

ttggagctca tcaagaaggg gtacacccag cagctggcct tcagacaacc cagctctgcc    180 

tttgcggcct tcctgaaccg ggcacccagc acctggctga ccgcctacgt ggtcaaggtc    240 

ttctctctgg ctgtcaacct cattgccatc gactcccagg tcctctgcgg ggctgttaaa    300 

tggctgatcc tggagaagca gaagcccgac ggggtcttcc aggaggatgc gcccgtgata    360 

catcaagaaa tgactggtgg attccggaac accaacgaga aagacatggc cctcacggcc    420 

tttgttctca tctcgctgca agaggctaaa gagatttgcg aggagcaggt caacagcctg    480 

cccggcagca tcactaaagc aggagacttc cttgaagcca actacatgaa cctacagaga    540 

tcctacactg tggccatcgc tgcctatgcc ctggcccaga tgggcaggct gaagggacct    600 

cttctcaaca aatttctgac cacagccaaa gataagaacc gctgggagga gcctggtcag    660 

cagctctaca atgtggaggc cacatcctat gccctcttgg ccctactgca gctaaaagac    720 

tttgactttg tgcctcccgt cgtgcgttgg ctcaatgaac agagatacta cggtggtggc    780 

tatggctcta cccaggccac cttcatggtg ttccaagcct tggctcaata ccaaaaggat    840 

gtccctgatc acaaggaact gaacctggat gtgtccctcc aactgccc                 888 

 
           
             26  
             888  
             DNA  
             Artificial Sequence  
             
               First variant Macaca mulatta C3d  
             
           
            26 

acgccaagcg gatcaggcga gcagaatatg atcactatga caccaaccgt aattgcggtc     60 

cattatctcg acgaaaccga acagtgggaa aaatttgggc cggaaaagcg tcaaggcgct    120 

ctcgagttga tcaagaaagg ctacacgcaa cagttagcgt tccgtcaacc atcatcagcg    180 

ttcgccgctt tcctgaatcg tgcgccatca acgtggctca cagcgtatgt agtgaaggta    240 

tttagcctcg ccgtaaattt aatcgcgatt gacagtcaag tgttatgcgg cgcggtcaag    300 

tggctcattc ttgaaaagca aaagccagat ggcgtattcc aagaggacgc cccagtcatc    360 

caccaagaga tgacaggcgg ctttcgcaat actaatgaga aggacatggc gttaaccgcg    420 

tttgtcttaa tcagtttaca ggaagccaaa gaaatttgtg aggaacaggt aaatagttta    480 

cctgggagta ttacgaaagc gggcgatttc ttagaagcaa attacatgaa tctccaacgc    540 

tcatacacgg tagcgatcgc ggcttatgcc ttagcgcaga tggggagatt aaaaggccca    600 

ttactgaaca agtttttaac aaccgcaaaa gacaagaata ggtgggagga accaggccaa    660 

caactttata acgtcgaagc aacgtcatac gcattattag cactcttaca actcaaggac    720 

ttcgacttcg taccacctgt ggtacggtgg cttaacgaac aaaggtatta cgggggcgga    780 

tacggcagca cgcaagcgac tttcatggtc tttcaagcac tcgcacagta ccagaaggat    840 

gttcctgatc acaaggaatt aaacttagat gtcagtctgc agttacca                 888 

 
           
             27  
             888  
             DNA  
             Artificial Sequence  
             
               Second variant Macaca mulatta C3d  
             
           
            27 

actccttcag ggagtggaga acaaaacatg attacaatga cccctacagt gatcgccgta     60 

cactacttag atgagacaga gcaatgggag aaattcggtc ccgagaaaag acagggagcg    120 

ttagaactta ttaaaaaggg atatacacag caactcgctt ttaggcagcc tagtagcgca    180 

tttgctgcgt ttctcaacag agcccctagt acatggttaa cggcttacgt cgtaaaagtg    240 

ttctcattag cggtgaacct gattgcaatc gattcgcagg tactgtgtgg agccgtgaaa    300 

tggttaatct tagagaaaca gaaacctgac ggagtgtttc aggaagatgc acctgtaatt    360 

caccaggaaa tgaccggggg cttcagaaac acaaacgaaa aagatatggc tctgacagct    420 

ttcgtgctga tttccctcca agaggcgaag gagatctgcg aagagcaagt gaactcactc    480 

ccaggatcaa tcaccaaggc cggggacttt ctggaggcga actatatgaa cttgcagagg    540 

agctataccg tcgcaattgc cgcatacgcg ctcgcacaaa tgggacgtct caaaggacct    600 

ctgttaaata aattcctcac gacggcgaag gataaaaacc gatgggaaga acctgggcaa    660 

cagttgtaca atgtagaggc gaccagttat gcgctgctcg cgttgctcca gttgaaagat    720 

tttgattttg tccctccagt agtcagatgg ttgaatgagc agcgttacta tggagggggg    780 

tatggatcaa cacaggcaac gtttatggta ttccaggcgt tagcgcaata tcaaaaagac    840 

gtgccagacc acaaagagct taatctcgac gtatcattac aactccct                 888 

 
           
             28  
             888  
             DNA  
             Artificial Sequence  
             
               Third variant Macaca mulatta C3d  
             
           
            28 

acaccgtctg gtagcggtga gcaaaatatg ataaccatga ctccgactgt tatagcagtt     60 

cactatttag acgagactga acaatgggaa aagtttggac cggaaaaaag gcaaggtgca    120 

ctggaattaa taaaaaaagg ttatacgcag caactagcgt tcaggcagcc gtccagcgct    180 

ttcgcagcat ttctgaacag ggctccgtcc acttggttga cggcatatgt cgtgaaagtt    240 

tttagtttgg cagttaactt gatagcgatc gatagccagg ttttgtgtgg tgcagtaaag    300 

tggttgatac tcgaaaagca aaagccggat ggtgtttttc aagaagacgc cccggttatc    360 

catcaggaga tgactggagg tttcaggaat accaatgaaa aggatatggc attgactgca    420 

ttcgtattga taagcttgca agaagcaaag gagatatgtg aagaacaagt taattccttg    480 

ccgggttcca taacaaaggc tggtgatttt ctcgaggcta attatatgaa tctgcaacga    540 

agttatacag ttgctatagc agcctacgca ctcgctcaaa tgggtcgctt gaagggtccg    600 

ctcctgaata agttcttgac tactgctaag gacaaaaata gatgggaaga gccgggacag    660 

caactgtata atgttgaagc tactagctac gctttgctgg ctctgttgca actgaaggat    720 

ttcgatttcg ttcccccggt tgttaggtgg ttaaacgagc aacgctatta tggcggaggt    780 

tacgggtcga ctcaagctac atttatggtt tttcaggctc tggcccagta tcagaaagat    840 

gtccccgatc ataaggagct caatctggac gttagcttgc agttgccg                 888 

 
           
             29  
             296  
             PRT  
             Macaca mulatta  
           
            29 

Thr Pro Ser Gly Ser Gly Glu Gln Asn Met Ile Thr Met Thr Pro Thr 
1               5                   10                  15 

Val Ile Ala Val His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe 
            20                  25                  30 

Gly Pro Glu Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr 
        35                  40                  45 

Thr Gln Gln Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala Phe 
    50                  55                  60 

Leu Asn Arg Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val 
65                  70                  75                  80 

Phe Ser Leu Ala Val Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys 
                85                  90                  95 

Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val 
            100                 105                 110 

Phe Gln Glu Asp Ala Pro Val Ile His Gln Glu Met Thr Gly Gly Phe 
        115                 120                 125 

Arg Asn Thr Asn Glu Lys Asp Met Ala Leu Thr Ala Phe Val Leu Ile 
    130                 135                 140 

Ser Leu Gln Glu Ala Lys Glu Ile Cys Glu Glu Gln Val Asn Ser Leu 
145                 150                 155                 160 

Pro Gly Ser Ile Thr Lys Ala Gly Asp Phe Leu Glu Ala Asn Tyr Met 
                165                 170                 175 

Asn Leu Gln Arg Ser Tyr Thr Val Ala Ile Ala Ala Tyr Ala Leu Ala 
            180                 185                 190 

Gln Met Gly Arg Leu Lys Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr 
        195                 200                 205 

Ala Lys Asp Lys Asn Arg Trp Glu Glu Pro Gly Gln Gln Leu Tyr Asn 
    210                 215                 220 

Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp 
225                 230                 235                 240 

Phe Asp Phe Val Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr 
                245                 250                 255 

Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln 
            260                 265                 270 

Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His Lys Glu Leu Asn 
        275                 280                 285 

Leu Asp Val Ser Leu Gln Leu Pro 
    290                 295 

 
           
             30  
             296  
             PRT  
             Macaca mulatta  
           
            30 

Thr Pro Ser Gly Ser Gly Glu Gln Asn Met Ile Thr Met Thr Pro Thr 
1               5                   10                  15 

Val Ile Ala Val His Tyr Leu Asp Glu Thr Glu Gln Trp Glu Lys Phe 
            20                  25                  30 

Gly Pro Glu Lys Arg Gln Gly Ala Leu Glu Leu Ile Lys Lys Gly Tyr 
        35                  40                  45 

Thr Gln Gln Leu Ala Phe Arg Gln Pro Ser Ser Ala Phe Ala Ala Phe 
    50                  55                  60 

Leu Asn Arg Ala Pro Ser Thr Trp Leu Thr Ala Tyr Val Val Lys Val 
65                  70                  75                  80 

Phe Ser Leu Ala Val Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys 
                85                  90                  95 

Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val 
            100                 105                 110 

Phe Gln Glu Asp Ala Pro Val Ile His Gln Glu Met Thr Gly Gly Phe 
        115                 120                 125 

Arg Asn Thr Asn Glu Lys Asp Met Ala Leu Thr Ala Phe Val Leu Ile 
    130                 135                 140 

Ser Leu Gln Glu Ala Lys Glu Ile Cys Glu Glu Gln Val Asn Ser Leu 
145                 150                 155                 160 

Pro Gly Ser Ile Thr Lys Ala Gly Asp Phe Leu Glu Ala Asn Tyr Met 
                165                 170                 175 

Asn Leu Gln Arg Ser Tyr Thr Val Ala Ile Ala Ala Tyr Ala Leu Ala 
            180                 185                 190 

Gln Met Gly Arg Leu Lys Gly Pro Leu Leu Asn Lys Phe Leu Thr Thr 
        195                 200                 205 

Ala Lys Asp Lys Asn Arg Trp Glu Glu Pro Gly Gln Gln Leu Tyr Asn 
    210                 215                 220 

Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu Gln Leu Lys Asp 
225                 230                 235                 240 

Phe Asp Phe Val Pro Pro Val Val Arg Trp Leu Asn Glu Gln Arg Tyr 
                245                 250                 255 

Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln 
            260                 265                 270 

Ala Leu Ala Gln Tyr Gln Lys Asp Val Pro Asp His Lys Glu Leu Asn 
        275                 280                 285 

Leu Asp Val Ser Leu Gln Leu Pro 
    290                 295