Abstract:
The present invention provides an isolated nucleic acid sequence encoding murine IL-2Rγ. The present invention also provides a vector comprising a mutated IL-2Rγ nucleic acid which is capable of homologous recombination in at least some cells to which the vector is introduced. The present invention also provides an embryonic stem cell comprising a mutated IL-2Rγ nucleic acid integrated into the cell by homologous recombination following transfection with the vector above. The present invention further provides a blastocyst cell comprising the embryonic stem cell above. In addition, the present invention provides a transgenic animal comprising a mutated IL-2Rγ gene. In particular, the animal is a non-human mammal whose germ and somatic cells contain a mutated IL-2Rγ gene sequence introduced into said mammal, or an ancestor thereof, at an embryonic stage. The present invention also provides a method of producing a non-human mammal with XSCID which comprises introducing into at least some cells of the recipient animal a mutated IL-2Rγ gene. Lastly, the present invention provides a non-human animal produced by the method above, and progeny thereof, wherein at least some cells retain a mutated IL-2Rγ gene.

Description:
This is a continuation of application Ser. No. 08/121,435, filed on Sep. 14, 1993, abandoned, which is a continuation-in-part application of U.S. Ser. No. 08/031,143, filed Mar. 12, 1993, the contents of which are hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     Severe combined immunodeficiency diseases (SCIDs) represent a spectrum of disorders characterized by profound defects of both cellular and humoral immunity (Cooper, M. D. and Butler, J. L. Fundamental Immunology, (Paul, W. E., editor, Raven Press, New York), pp. 1034-1039 (1989); Gelfand, E. W. and Dosch, H. M. Birth Defects: Original Article Series 19(3): 65-72 (1983); Conley, M. E. Annu. Rev. Immunol. 10: 215-238 (1992)). One in every 10 5  to 10 6  live births are affected by these diseases. Infants with SCID usually become ill in the first few months of life. While their growth and development may initially proceed normally, infections leading to cessation of growth soon become evident (Cooper, M. D. and Butler, J. L., supra, at 1034). Individuals with SCID are vulnerable to virtually every type of pathogenic microorganism, even those that rarely cause disease in normal individuals (Cooper, M. D. and Butler, J. L., supra, at 1034). Candida fungal infection of mucocutaneous surfaces is often the first indication of immunodeficiency, followed by intractable diarrhea and pneumonia (Cooper, M. D. and Butler, J. L., supra, at 1034). The majority of infected infants die before their first birthday. 
     Classical SCID (&#34;Swiss-type agammaglobulinemia&#34;) is characterized by the absence of both T and B cells, presumably related to a defect affecting the lymphocytic stem cell. Autosomal recessive forms of SCID result from deficiencies of adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP), the inability to express class II molecules of the major histocompability complex (&#34;Bare Lymphocyte Syndrome&#34;), or defective IL-2 production. Other autosomal recessive forms have no known defect (Cooper, M. D. and Butler, J. L., supra, at 1034-1037; Gelfand, E. W. and Dosch, H. M., supra, at 66-67; Conley, supra, at 215-238). 
     X-linked severe combined immunodeficiency (XSCID) accounts for approximately half of all cases of SCID. This form of SCID is inherited in an X-linked fashion. XSCID is characterized by an absence of T-cells and histologic evidence of hypoplastic and abnormal differentiation of the thymic epithelium. Levels of B-cells are normal or even elevated, and therefore patients are only mildly lymphopenic (Cooper, M. D. and Butler, J. L., supra, at 1037; Gelfand, E. W. and Dosch, H. M., supra, at 66-70; Conley, M. E., supra, at 226-227). Since the B-cells are not functional, these males are hypo- or agammaglobulinemic. 
     In U.S. Ser. No. 08/031,143, filed Mar. 12, 1993, U.S Pat. No. 5,518,880 and in Noguchi, M., et al. Cell 73: 147-157 (1993), it was determined that XSCID results from a defective or mutated IL-2Rγ gene. 
     Human IL-2Rα (Leonard, W. J., et al. Nature (London) 311: 625-631 (1984); Nikaido, T., et al. Nature (London) 311: 631-635 (1984)), IL-2Rβ (Hatakcyama, M., et al. Science 244: 551-556 (1989)), and IL-2Rγ (Takeshita, T., et al. Science 257: 379-382 (1992)) cDNAs have been isolated. Murine cDNAs, however, have only been isolated for IL-2Rα (Miller, J., et al. J. Immunol. 134: 4212-4217 (1985); Shimuzu, A., et al. Nucleic Acids Res. 13: 1505-1516 (1985)) and IL-2Rβ (Kono, T., et al. Proc. Natl. Acad. Sci. USA 87: 1806-1810 (1990)). 
     The present invention is based upon the isolation of murine IL-2Rγ cDNA, and its uses thereof. One important application of the murine IL-2Rγ cDNA is the preparation of an IL-2Rγ deficient mouse. The IL-2Rγ deficient mouse should serve as an excellent animal model for XSCID. 
     SUMMARY OF THE INVENTION 
     The present invention provides an isolated nucleic acid sequence encoding murine IL-2Rγ. 
     The present invention also provides a vector comprising a mutated IL-2Rγ nucleic acid which is capable of homologous recombination in at least some cells to which the vector is introduced. 
     The present invention also provides an embryonic stem cell comprising a mutated IL-2Rγ nucleic acid integrated into the cell by homologous recombination following transfection with the vector above. 
     The present invention further provides a blastocyst cell comprising the embryonic stem cell above. 
     In addition, the present invention provides a transgenic animal comprising a mutated IL-2Rγ gene. In particular, the animal is a non-human mammal whose germ and somatic cells contain a mutated IL-2Rγ gene sequence introduced into said mammal, or an ancestor thereof, at an embryonic stage. 
     The present invention also provides a method of producing a non-human mammal with XSCID which comprises introducing into at least some cells of the recipient animal a mutated IL-2Rγ gene. 
     Lastly, the present invention provides a nonhuman animal produced by the method above, and progeny thereof, wherein at least some cells retain a mutated IL-2Rγ gene. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1. DNA and deduced amino acid sequence for murine IL-2Rγ (SEQ. ID. NOS. 1 and 2) The putative signal peptide and transmembrane domain are in italics; the transmembrane domain is additionally underscored with a heavy bar. The four conserved cysteines and the WSXWS motif are boxed. The ATG start codon, TGA stop codon, and AATAAA polyadenylylation signal are underlined. Each N-linked glycosylation consensus motif (Asn-X-Ser/Thr) is double underlined. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides an isolated nucleic acid sequence encoding murine IL-2Rγ. In the preferred embodiment, the nucleic acid sequence is selected from the group consisting of: (a) The nucleic acid sequence contained in FIG. 1, or a complementary strand thereof; (b) DNA sequences which hybridize to the DNA sequence defined in (a); and (c) DNA sequences, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) or (b). The present invention also provides a murine IL-2Rγ protein encoded by the isolated nucleic acid sequence above. Preferably, the protein has the amino acid sequence contained in FIG. 1. 
     The cDNA encoding the murine IL-2Rγ was isolated and characterized from a library prepared from mRNA from Con A activated splenocytes from CBA/Ca mice. The full length murine IL-2Rγ cDNA is 1608 bp with an open reading frame that encodes 369 amino acids, identical in length to the open reading frame of the human IL-2Rγ cDNA. Murine and human IL-2Rγ have 69 and 70% identity at the nucleotide and amino acid levels, respectively. The murine IL-2Rγ chain retains the WSXWS motif and four cysteine residues characteristic of cytokine receptor superfamily members (Bazan, J. F. Proc. Natl. Acad. Sci. USA 87: 6934-6938 (1990)). Six N-linked glycosylation sites (asn-X-ser/thr) are found in the extracellular domain. Although the human IL-2Rγ cytoplasmic domain has a region of limited homology to the fourth or fifth Src homology region 2 (SH2) subdomains (Takeshita, et al., supra), this region is less well conserved in mouse (in particular, arg 289 and thr 292 in human IL-2Rγ are conserved in Lck, Hck, Lyn, and Blk, but not in murine IL-2Rγ). A &#34;leucine zipper&#34; like motif was noted to exist in the human IL-2Rγ sequence (formed by leucines at residues 165, 172, 179, and 186)(Takeshita, et al., supra), although no clear functional role for this was demonstrated. In the murine sequence, leucines 165 and 172 are conserved, leucine 179 is replaced by isoleucine and the final leucine is at position 187 instead of 186. The lack of rigorous conservation in the murine sequence of the limited SH2 subdomain homology and leucine zipper like regions lessens the probability that they play critical roles. 
     The murine IL-2Rγ CDNA is preferably used to prepare a transgenic mouse containing a mutated IL-2Rγ CDNA. This mouse would be beneficial for studying XSCID, which results from a defective or mutated IL-2Rγ gene (see U.S. Ser. No. 08/031,143, filed Mar. 12, 1993U.S. Pat. No. 5,518,880) where it was shown that three patients with XSCID had different point mutations resulting in premature stop codons at lys 97, arg 267, and ser 286, respectively, resulting in truncations of 251, 81, and 62 amino acids, respectively, and wherein it was taught that other types of point mutations, such as those which affect residues required for IL-2 binding, would also be expected to be found if DNA from enough XSCID patients were sequenced. 
     A transgenic mouse may be prepared by homologous recombination techniques (see Current Protocols in Molecular Biology, edited by F. M. Ausubel, et al., Supp. 23, §IV, pp. 9.15-9.17 (1993)). The vectors used for homologous recombination may be insertion constructs or replacement constructs. 
     The insertion construct contains a region of homology to the target gene cloned as a single continuous sequence and is linearized by cleavage of a unique restriction site within the region of homology. Homologous recombination introduces the insertion construct sequences into the homologous site of the target gene, interrupting normal target-gene structure by adding sequences. As a result, the normal gene can be regenerated from the mutated target gene by an intrachromosomal recombination event. (see Current Protocols in Molecular Biology, supra, p. 9.15.1). 
     The replacement construct is more commonly used. It contains two regions of homology to the target gene located on either side of a mutation, which is usually a positive selectable marker. Homologous recombination proceeds by a double cross-over event that replaces the target-gene sequences with the replacement-construct sequences. Because no duplication of sequences occurs, the normal gene cannot be regenerated. (see Current Protocols in Molecular Biology, supra, p. 9.15.1). 
     Homologous recombination may be used to inactivate a gene completely (&#34;knock out&#34;) by creating a deletion in part of the gene or by deleting the entire gene. Usually, the construct contains a target gene with a portion replaced with a drug-resistant gene such as neomycin. (see Current Protocols in Molecular Biology, supra, p. 9.15.3). 
     Homologous recombination may also be used to introduce subtle mutations (e.g. single point mutations). One known method is called the &#34;hit and run&#34; method (Hasty, et al., Nature 350: 243-246 (1991)). In the Hasty, et al. method, an insertion vector is used to introduce a duplication containing a mutation into the target gene. One copy of the duplicated region is removed under gancyclovir selection. Fifty percent of the time it will be the normal half, leaving the mutation. (see Current Protocols in Molecular Biology, supra, p. 9.15.4). 
     In the preferred embodiment, the vector pPNT (Tybulewicz, et al., Cell 65: 1153-1163 (1991)) is used to make a targeting IL-2Rγ construct. pPNT is a &#34;replacement&#34; (as opposed to &#34;insertion&#34;) vector in which two fragments for homologous recombination flank the neomycin resistance gene. Expression of the neomycin resistance gene is driven by the PGK promoter. The neomycin resistance gene allows for positive selection. Downstream of the second fragment cloning site is the HSV thymidine kinase gene, also driven by the PGK promoter, which allows for negative selection. The procedures using the pPNT vector are well known and are described in Current Protocols in Molecular Biology, supra, pp. 9.16.1-9.16.9. 
     As an example of the subject invention, two IL-2Rγ genomic fragments of the murine IL-2Rγ which do not contain repetitive sequences were cloned into pPNT. The plasmid was linearized with Not 1 and transfected into J1 embryonic stem cells (gift of R. Jaenisch, MIT) derived from 129 strain mice (i.e., the same strain as the genomic library in order to maximize the sequence identity for efficient homologous recombination). 
     G418 and gancyclovir were added 24 hours following transfection to effect &#34;positive-negative selection&#34; in which G418 is used to positively select colonies containing the neomycin resistance gene and gancyclovir is used to eliminate random integration. In random integration, the flanking TK gene is retained and confers gancyclovir sensitivity, whereas in homologous recombination the TK gene will be lost due to cross-over and the cells will be gancyclovir resistant. Gancyclovir selection also helps to eliminate multiple copies of the DNA fragments so that clones with single integrated fragments were obtained. 
     The clones are then analyzed for homologous recombination by Southern blot and/or PCR making use of sequences flanking the fragments used for homologous recombination to confirm that the site of integration is indeed at the IL-2Rγ locus rather than at a random locus. 
     The IL-2Rγ deficient ES cells are then infected into blastocysts and subsequently implanted into female mice by known procedures. It should be noted that J1 ES cells are of male lineage and therefore contain only a single X chromosome. As a result, only a single &#34;knockout&#34; is required to make an IL-2Rγ deficient ES cell. After IL-2Rγ deficient ES cells are injected into blastocysts, the initial mice obtained will be healthy chimeras. The germline will have both normal and mutated X chromosome bearing cells depending on whether they are derived from the normal blastocyst derived cells or the mutant ES cells. Breeding will yield heterozygous females who then have the possibility of having IL-2Rγ deficient male offspring. It is possible that these males will be severely immunodeficient. However, given the ability to breed the mice in microisolator sterile environments, they may be able to survive if shielded from infection. 
     Transgenic mice also may be prepared by using the technique of recombination activating gene (RAG)-2-deficient blastocyst complementation (Chen, et al., Proc. Natl. Acad. Sci. 90: 4228-4232 (1993)). The principle of this method is that RAG-2 deficient blastocysts generate chimeras with mature B and T cells which derive from the injected ES cells. This method offers the ability to create an IL-2Rγ deficient ES cell which can be complemented with IL-2Rγ constructs containing subtle mutations whose functional significance is to be evaluated. In this fashion, multiple different IL-2Rγ constructs may be injected into IL-2Rγ deficient ES cells and progeny mice studied far faster than multiple transgenic lines could be established for mating. 
     All publications mentioned hereinabove are hereby incorporated by reference in their entirety. 
     While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that the invention should not be construed to be limited as such, and that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- (1) GENERAL INFORMATION:-    (iii) NUMBER OF SEQUENCES:  2- (2) INFORMATION FOR SEQ ID NO:1:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH:  1608     (B) TYPE:  NUCLEIC A - #CID     (C) STRANDEDNESS:  SING - #LE     (D) TOPOLOGY:  UNKNOWN-     (ii) MOLECULE TYPE:     (A) DESCRIPTION:  OLIGO - #NUCLEOTIDE-    (iii) HYPOTHETICAL:  NO-     (iv) ANTI-SENSE:  NO-     (vi) ORIGINAL SOURCE:     (A) ORGANISM:  MURINE# IL-2R   (C) INDIVIDUAL ISOLATE:-     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:1:- CCCAGAGAAA GAAGAGCAAG CACC ATG TTG AAA CTA TTA T - #TG TCA CCT  48Met Leu Lys Leu Leu Leu Ser Pro1               5- AGA TCC TTC TTA GTC CTT CAG CTG CTC CTG CT - #G AGG GCA GGG#  90Arg Ser Phe Leu Val Leu Gln Leu Leu Leu Le - #u Arg Ala Gly#     20- TGG AGC TCC AAG GTC CTC ATG TCC AGT GCG AA - #T GAA GAC ATC# 132Trp Ser Ser Lys Val Leu Met Ser Ser Ala As - #n Glu Asp Ile#         35- AAA GCT GAT TTG ATC CTG ACT TCT ACA GCC CC - #T GAA CAC CTC# 174Lys Ala Asp Leu Ile Leu Thr Ser Thr Ala Pr - #o Glu His Leu#             50- AGT GCT CCC ACT CTG CCC CTT CCA GAG GTT CA - #G TGC TTT GTG# 216Ser Ala Pro Thr Leu Pro Leu Pro Glu Val Gl - #n Cys Phe Val#                 60- TTC AAC ATA GAG TAC ATG AAT TGC ACT TGG AA - #T AGC AGT TCT# 258Phe Asn Ile Glu Tyr Met Asn Cys Thr Trp As - #n Ser Ser Ser# 75- GAG CCT CAG GCA ACC AAC CTC ACG CTG CAC TA - #T AGG TAC AAG# 300Glu Pro Gln Ala Thr Asn Leu Thr Leu His Ty - #r Arg Tyr Lys#     90- GTA TCT GAT AAT AAT ACA TTC CAG GAG TGC AG - #T CAC TAT TTG# 342Val Ser Asp Asn Asn Thr Phe Gln Glu Cys Se - #r His Tyr Leu#        105- TTC TCC AAA GAG ATT ACT TCT GGC TGT CAG AT - #A CAA AAA GAA# 384Phe Ser Lys Glu Ile Thr Ser Gly Cys Gln Il - #e Gln Lys Glu#           120- GAT ATC CAG CTC TAC CAG ACA TTT GTT GTC CA - #G CTC CAG GAC# 426Asp Ile Gln Leu Tyr Gln Thr Phe Val Val Gl - #n Leu Gln Asp#               130- CCC CAG AAA CCC CAG AGG CGA GCT GTA CAG AA - #G CTA AAC CTA# 468Pro Gln Lys Pro Gln Arg Arg Ala Val Gln Ly - #s Leu Asn Leu135                 1 - #40                 1 - #45- CAG AAT CTT GTG ATC CCA CGG GCT CCA GAA AA - #T CTA ACA CTC# 510Gln Asn Leu Val Ile Pro Arg Ala Pro Glu As - #n Leu Thr Leu#   160- AGC AAT CTG AGT GAA TCC CAG CTA GAG CTG AG - #A TGG AAA AGC# 552Ser Asn Leu Ser Glu Ser Gln Leu Glu Leu Ar - #g Trp Lys Ser#       175- AGA CAT ATT AAA GAA CGC TGT TTA CAA TAC TT - #G GTG CAG TAC# 594Arg His Ile Lys Glu Arg Cys Leu Gln Tyr Le - #u Val Gln Tyr#           190- CGG AGC AAC AGA GAT CGA AGC TGG ACG GAA CT - #A ATA GTG AAT# 636Arg Ser Asn Arg Asp Arg Ser Trp Thr Glu Le - #u Ile Val Asn#               200- CAT GAA CCT AGA TTC TCC CTG CCT AGT GTG GA - #T GAC CTG AAA# 678His Glu Pro Arg Phe Ser Leu Pro Ser Val As - #p Glu Leu Lys205                 2 - #10                 2 - #15- CGG TAC ACA TTT CGG GTT CGG AGC CGC TAT AA - #C CCA ATC TGT# 720Arg Tyr Thr Phe Arg Val Arg Ser Arg Tyr As - #n Pro Ile Cys#   230- GGA AGT TCT CAA CAG TGG AGT AAA TGG AGC CA - #G CCT GTC CAC# 762Gly Ser Ser Gln Gln Trp Ser Lys Trp Ser Gl - #n Pro Val His#       245- TGG GGG AGT CAT ACT GTA GAG GAG AAT CCT TC - #C TTG TTT GCA# 804Trp Gly Ser His Thr Val Glu Glu Asn Pro Se - #r Leu Phe Ala#           260- CTG GAA GCT GTG CTT ATC CCT GTT GGC ACC AT - #G GGG TTG ATT# 846Leu Glu Ala Val Leu Ile Pro Val Gly Thr Me - #t Gly Leu Ile#               270- ATT ACC CTG ATC TTT GTG TAC TGT TGG TTG GA - #A CGA ATG CCT# 888Ile Thr Leu Ile Phe Val Tyr Cys Trp Leu Gl - #u Arg Met Pro275                 2 - #80                 2 - #85- CCA ATT CCC CCC ATC AAG AAT CTA GAG GAT CT - #G GTT ACT GAA# 930Pro Ile Pro Pro Ile Lys Asn Leu Glu Asp Le - #u Val Thr Glu#   300- TAC CAA GGG AAC TTT TCC GCC TGG AGT GGT GT - #G TCT AAA GGG# 972Tyr Gln Gly Asn Phe Ser Ala Trp Ser Gly Va - #l Ser Lys Gly#       315- CTG ACT GAG AGT CTG CAG CCA GAC TAC AGT GA - #A CGG TTC TGC#1014Leu Thr Glu Ser Leu Gln Pro Asp Tyr Ser Gl - #u Arg Phe Cys#           330- CAG GTC AGC GAG ATT CCC CCC AAA GGA GGG GC - #C CTA GGA GAG#1056His Val Ser Glu Ile Pro Pro Lys Gly Gly Al - #a Leu Gly Glu#               340- GGG CCT GGA GGT TCT CCT TGC AGC CTG CAT AG - #C CCT TAC TGG#1098Gly Pro Gly Gly Ser Pro Cys Ser Leu His Se - #r Pro Tyr Trp345                 3 - #50                 3 - #55- CCT CCC CCA TGT TAT TCT CTG AAG CCG GAA GC - #C TGAACATCAA#1141Pro Pro Pro Cys Tyr Ser Leu Lys Pro Glu Al - #a#   365#            1191CTCAAA GTCCTATAGT CCTAAGTGAC GCTAACCTCC#            1241CAATCT GGATCCAATG CTCACTGCCT TCCCTTGGGG#            1291CCTGTC CCATGTAACT GCCTTTCTGT TCCATATGCC#            1341CCCTTG CCCTCTTTCC CTGCACAAGC CCTCCCATGC#            1391TTCCAC TTTCTTTGAA GAGAGTCTTA CCCTGTAGCC#            1441GCTCAC TATGTAGCCA GGTTGGCCTC CAACTCACAG#            1491TCTGCC TCATAAGAGT TGGGGTTACT GGCATGCACC#            1541GGTCCT TCTCTTTTAT AGGATTCTCC CTCCCTTTTT#            1591ACTGTT TCCAAATCAA CAAGAAATAA AGTTTTTAAC# 1608             A- (2) INFORMATION FOR SEQ ID NO:2:-      (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH:  369     (B) TYPE:  AMINO ACI - #D     (D) TOPOLOGY:  UNKNOWN-     (ii) MOLECULE TYPE:     (A) DESCRIPTION:  PROTE - #IN-    (iii) HYPOTHETICAL:  NO-     (vi) ORIGINAL SOURCE:     (A) ORGANISM:  MURINE# IL-2R   (C) INDIVIDUAL ISOLATE:-     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:2:- Met Leu Lys Leu Leu Leu Ser Pro Arg Ser Ph - #e Leu Val Leu Gln#                15- Leu Leu Leu Leu Arg Ala Gly Trp Ser Ser Ly - #s Val Leu Met Ser#                30- Ser Ala Asn Glu Asp Ile Lys Ala Asp Leu Il - #e Leu Thr Ser Thr#                45- Ala Pro Glu His Leu Ser Ala Pro Thr Leu Pr - #o Leu Pro Glu Val#                60- Gln Cys Phe Val Phe Asn Ile Glu Tyr Met As - #n Cys Thr Trp Asn#                75- Ser Ser Ser Glu Pro Gln Ala Thr Asn Leu Th - #r Leu His Tyr Arg#                90- Tyr Lys Val Ser Asp Asn Asn Thr Phe Gln Gl - #u Cys Ser His Tyr#                105- Leu Phe Ser Lys Glu Ile Thr Ser Gly Cys Gl - #n Ile Gln Lys Glu#               120- Asp Ile Gln Leu Tyr Gln Thr Phe Val Val Gl - #n Leu Gln Asp Pro#               135- Gln Lys Pro Gln Arg Arg Ala Val Gln Lys Le - #u Asn Leu Gln Asn#               150- Leu Val Ile Pro Arg Ala Pro Glu Asn Leu Th - #r Leu Ser Asn Leu#               165- Ser Glu Ser Gln Leu Glu Leu Arg Trp Lys Se - #r Arg His Ile Lys#               180- Glu Arg Cys Leu Gln Tyr Leu Val Gln Tyr Ar - #g Ser Asn Arg Asp#               195- Arg Ser Trp Thr Glu Leu Ile Val Asn His Gl - #u Pro Arg Phe Ser#               210- Leu Pro Ser Val Asp Glu Leu Lys Arg Tyr Th - #r Phe Arg Val Arg#               225- Ser Arg Tyr Asn Pro Ile Cys Gly Ser Ser Gl - #n Gln Trp Ser Lys#               240- Trp Ser Gln Pro Val His Trp Gly Ser His Th - #r Val Glu Glu Asn#               255- Pro Ser Leu Phe Ala Leu Glu Ala Val Leu Il - #e Pro Val Gly Thr#               270- Met Gly Leu Ile Ile Thr Leu Ile Phe Val Ty - #r Cys Trp Leu Glu#               285- Arg Met Pro Pro Ile Pro Pro Ile Lys Asn Le - #u Glu Asp Leu Val#               300- Thr Glu Tyr Gln Gly Asn Phe Ser Ala Trp Se - #r Gly Val Ser Lys#               315- Gly Leu Thr Glu Ser Leu Gln Pro Asp Tyr Se - #r Glu Arg Phe Cys#               330- His Val Ser Glu Ile Pro Pro Lys Gly Gly Al - #a Leu Gly Glu Gly#               345- Pro Gly Gly Ser Pro Cys Ser Leu His Ser Pr - #o Tyr Trp Pro Pro#               360- Pro Cys Tyr Ser Leu Lys Pro Glu Ala           365__________________________________________________________________________