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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/10—RNA viruses
  • C07K16/112—Retroviridae (F), e.g. leukemia viruses
  • C07K16/114—Lentivirus (G), e.g. human immunodeficiency virus [HIV], feline immunodeficiency virus [FIV] or simian immunodeficiency virus [SIV]
  • C07K16/1145—Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
  • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• Polypeptide fragment capable of inducing neutralising antibodies against Feline Immuno-deficiency virus.
• the present invention is concerned with a polypeptide fragment of the Feline Immuno-deficiency virus surface protein, i munogen ⁇ comprising the polypeptide fragment, a nucleic acid sequence encoding the polypeptide fragment, a recombinant nucleic acid molecule containing the nucleic acid sequence, virus vectors containing the nucleic acid sequence or the recombinant nucleic acid molecule, host cells containing that nucleic acid sequence or the recombinant nucleic acid molecules or the vector virus, a vaccine for the protection of cats against Feline Immuno-deficiency virus, monoclonal antibodies reactive with the polypeptide fragment, and the use of the polypeptide or the immunogen for the preparation of a vaccine against Feline Immuno-deficiency virus.
• FMV Feline Immuno-deficiency virus
• FIV-infection in cats may lead to immunological abnormalities similar to those seen in Human Immuno- deficiency virus type 1 (HIV-1) infected humans, like a depletion of CD4 + cells in the circulation.
• HV-1 Human Immuno- deficiency virus type 1
• PBMC peripheral blood mononuclear cells
• the pathogenesis is much alike HIV-1 pathogenesis: cats, experimentally infected with FIV appear normal for about 4-6 weeks. At that time they develop a low-grade fever, neutropenia and mild leukopenia, and generalised lymphadenopathy. This lymphadenopathy may persist up to 9 months. After this period, most animals are completely recovered from initial infection. After one year or more after initial infection, the onset of the terminal AIDS-like phase may take place.
• Lentiviruses by nature do display a large degree of molecular and biological variation. This natural variation is generally ascribed to the low fidelity of the viral enzyme reverse transcriptase in the process of copying the viral genomic RNA to DNA (Preston et al, Science 242: 1168-1171 (1988), Roberts et al, Science 242: 1171-1173 (1988)). As a result, several variant FIV-strains have been found.
• subunit-based vaccines as described in the present application has a number of significant advantages over the use of whole virus vaccines: a) There is no need for culturing live virus. This eliminates the introduction of unwanted mutations leading to more immunologically variant strains during virus growth.
• the occurrence of mutations in RNA- viruses is known to be high compared to DNA-viruses.
• retroviruses like e.g. FIV, the mutation rate is even higher, due to the high error rate of reverse transcriptase.
• escape mutants in vivo, (viruses, not recognized by their host's defenses) , and the role of antibody escape in viral persistance have recently been described by Pancino et al (Journ.
• live attenuated virus poses the problem of how to create a sufficiently attenuated vaccine, especially for the following reason: infected cats, especially in a later stage of infection may be immuno-impaired, and as a result, would suffer from severe illness, due to vaccination with even a highly attenuated live vaccine. (Gardner et al, Veterinary Medicine vol. march; 300-307. (1991)). Aiming at the subunit approach, research efforts are mainly aimed at the localisation of the immunologically important immunogenic determinants, the so-called epitopes at the FIV proteins.
• polypeptides synthesized either chemically or in prokaryotic expression systems are used.
• the present invention provides a polypeptide fragment of the Feline Immuno-deficiency Virus surface protein, characterised in that the polypeptide fragment comprises an amino acid sequence given in SEQ ID NO: 4 or a portion thereof capable of inducing neutralising antibodies against Feline Immuno-deficiency Virus.
• the fragment given in SEQ ID NO: 4 may also be referred to as the Central Fragment.
• the fragment in SEQ ID NO: 4 is a part of the FIV- protein shown in SEQ ID NO: 2.
• the present invention also provides a polypeptide fragment of the Feline Immuno-deficiency Virus surface protein, characterised in that the polypeptide fragment comprises an epitope located in the amino acid sequence given in SEQ ID NO: 4, capable of inducing antibodies that competitively inhibit binding of the neutralising monoclonal antibody from hybridoma 1E1EB4-93030567 as deposited with the European Collection of Animal Cell Cultures (further referred to as the ECACC) , Division of Biology. Salisbury, Wiltshire, SP4 OJG, United Kingdom, to native surface protein.
• ECACC European Collection of Animal Cell Cultures
• polypeptide fragment refers to an amino acid sub-set of the amino acid sequence representing the surface protein, comprising amino acids 361 to 445, the Central Fragment, or a portion thereof, and described in SEQ ID NO: 4.
• portion here refers to a subset of the amino acids represented by the SEQ ID NO: 4.
• Variation may be the result of insertion or deletion of one or more amino acids, or of replacement of one or more amino acids by functional equivalents. Replacement by functionally equivalents is often seen. Examples described by Neurath et al (The Proteins, Academic Press, New York (1979), page 14, figure 6) are i.a. the replacement of the amino acid alanine by serine; Ala/Ser, or Val/Ile, Asp/Glu, etc. In addition to the variations mentioned above, variations have been found, in which an amino acid has been replaced by another amino acid that is not a functional equivalent. This kind of variation only differs from replacement with functional equivalents in that it may yield a protein that has a slight modification in its spacial folding.
• epitope refers to an amino acid sequence containing at least 8 amino acid sequences, and capable of inducing (with or without flanking amino acids or immunostimulatory compounds) an immunological reaction in a suitable host animal (Geysen, et al; Proc. Natl. Acad. Sci. USA 81: 3998- 4002 (1984)).
• epitope may also span a polypeptide fragment larger than 8 amino acids, also depending on the epitope 1 s conformational nature.
• epitope in this context may refer to any amino acid sequence equal to, or larger than 8 amino acids.
• immunogenic refers to amino acid sequences capable of triggering the immune system.
• immuno-dominant region refers to an amino acid sequence that is capable of inducing a more than significant antibody response. This induction may result in relatively large amounts of antibodies directed against one single epitope, or in antibodies directed against a number of epitopes within this region.
• Neutralizing antibodies are antibodies capable of preventing the virus from multiplication in the host, thus interfering with the process of pathogenesis in such a manner, that the process of pathogenesis is inhibited.
• the role of neutralising antibodies has i.a. been described extensively by Fazekas de St. Groth (The neutralisation of viruses; Advances in Virus Research 9: 1-125 (1962))
• This can be done e.g. with the use of protein-digesting enzymes, e.g. proteinase K and V8- protease.
• protein-digesting enzymes e.g. proteinase K and V8- protease.
• the available amino acid information to synthesize the desired polypeptide fragment by using chemical synthesis. In that case, every unwanted amino acid sequence can be deliberately left out.
• One often used method for the chemical synthesis of short polypeptides is the Merrifield synthesis (Merrifield et al; J. Am. Che . Soc. 85:2149 (1963)).
• Merrifield synthesis Merrifield synthesis
• .Another way of synthesizing the polypeptide fragment is to clone the rDNA coding for the polypeptide into an expression vector and to express the genetic information in a suitable expression system. This possibility is described in detail below.
• the polypeptide fragment in a preferred form is a portion of the central fragment and said portion comprises at least an epitope located in between amino acid 389 and amino acid 412, or an epitope reactive with monoclonal antibody from hybridoma 1E1EB4-93030567 deposited with the ECACC.
• the portion of the polypeptide fragment is selected from the group of sequences comprising SEQ ID NO: 5, 6, 7.
• the invention also relates to an immunogen comprising a polypeptide fragment according to the present invention, linked to a carrier.
• carrier applies to molecules that are covalently linked to a polypeptide fragment of the invention and as such "carry” the polypeptide fragment.
• immunogen here refers to a polypeptide fragment of the present invention, presented to the immune system of a suitable host in such a form that it is capable of inducing an immunological response. It is well known to those skilled in the art, that the immunogenicity of polypeptides may be significantly enhanced by adding, or linking other molecules to a polypeptide of the present invention.
• polypeptides e.g. polypeptides with a length of 8 amino acids, are not immunogenic as such.
• these short polypeptides may be linked in various ways to other molecules, so-called carrier- molecules.
• carrier-molecules may e.g. be polypeptides.
• One way of making such an immunogen is the use of chemical methods to link a polypeptide fragment of the present invention to a carrier-protein.
• Proteins often used as carriers are e.g. Keyhole Limpet Haemocyanine and Bovine Serum Albumin.
• Methods of chemical linkage of polypeptides are i.a. described by Reichlin, M. ; Methods in Enzymology 70: 159-165 (1980) and by Erlanger, B.F.; Methods in Enzymology 70: 85-103 (1980) .
• Another way of making such an immunogen is the molecular cloning of the nucleotide sequence coding for a polypeptide of the present invention upstream, downstream or in between nucleotide sequences coding for another protein. Expression of this construct will then lead to a larger polypeptide, in which the polypeptide of the present invention is preceded, flanked or followed by other polypeptide sequences. Suitable flanking sequences could be those, coding for KLH or BSA, but many other protein sequences would be applicable as well.
• .Another suitable group of carrier molecules is the group comprising the complex carbohydrates. It is possible to chemically link a polypeptide of the present invention to a carbohydrate with the aim of enhancing the im uno-reactivity of the thus formed complex. Methods for covalent linkage of polypeptides to carbohydrates have been described a.o. by Dick, W.E. and Beurt, M. ; Contrib. Microbiol. Immunol. 10:48-114 (1989).
• the carrier is selected from the group of carriers consisting of surface-active compounds, sugars and proteins.
• the invention also provides a nucleic acid sequence encoding a polypeptide fragment or the immunogen according to the present invention.
• the amino acid building blocks of the polypeptide each have a corresponding nucleic acid triplet coding for that specific amino acid. This does not mean, however, that a single amino acid also has one single nucleic acid triplet coding for it. On the contrary, most amino acids have two to even six (Leucine) possible coding nucleic acid triplets. This phenomenon is known as the degeneracy of the genetic code.
• nucleic acid sequence leading to different but functionally homologous amino acids are also considered to be within the scope of this invention.
• said nucleic acid sequence comprises at least part of the nucleic acid sequence shown in SEQ ID NO: 3.
• said nucleic acid sequence is part of a recombinant nucleic acid molecule comprising the nucleic acid sequence under the control of regulating sequences enabling expression of the protein encoded by said nucleic acid sequence.
• Regulating sequences enabling expression of genes or fragments of genes may e.g. be promotor-sequences either or not in combination with enhancer sequences.
• Promotor sites are sequences to which RNA polymerase binds, initial to transcription.
• Promotor-sites exist in a variety of types, a.o. depending on the type of cell, they originate from. Promotor sequences have been described for promoters from prokaryotic, eukaryotic, and viral origin.
• Recombinant DNA molecules of the above mentioned type can be made by cutting a suitable DNA fragment with a suitable restriction enzyme, cutting a fragment containing regulating sequences with the same enzyme and ligating both fragments in such a way, that the nucleic acid sequence to be expressed is under the control of the promotor sequence.
• recombinant nucleic acid sequences will be cloned into a vector molecule.
• the then formed recombinant vector molecule often capable of self- replication in a suitable host cell, can be used to bring the cloned nucleic acid sequences into a cell.
• This may be a cell in which replication of the recombinant vector molecule occurs. It may also be a cell in which a regulating sequence of the vector is recognised, so that a polypeptide fragment according to the present invention is expressed.
• vectors for use in bacteria e.g. pBR322, 325 and 328, various pUC-vectors a.o. pUC 8, 9, 18, 19, specific expression-vectors; pGEM, pGEX, and Bluescript (R) , vectors based on bacteriophages; lambda-gtWes, Charon 28, M13-derived phages, vectors containing viral sequences on the basis of SV40, papilloma-virus, adenovirus or polyomavirus (Rodriguez, R.L. and Denhardt, D.T., ed. ; Vectors: A survey of molecular cloning vectors and their uses, Butterworths (1988), Lenstra et al. Arch. Virol.; 110: 1-24 (1990)).
• nucleic acid sequence under the control of regulating sequences enabling expression of the protein encoded by said nucleic acid sequence are considered to be part of the present invention.
• the nucleic acid sequence coding for a polypeptide, according to the present invention may be cloned either or not under the control of a promotor sequence, in a viral genome.
• the virus may be used as a way of transporting the nucleic acid sequence into a target cell.
• Such recombinant viruses are called vector viruses.
• the site of integration may be a site in a gene, not essential to the virus, or a site in an intergenic region.
• Viruses often used as vectors are Vaccinia viruses (Panicali et al; Proc. Natl. Acad. Sci. USA, 79: 4927 (1982), Herpesviruses (E.P.A.
• Retroviruses Valerio, D. et al; in Baum, S.J., Dicke, K.A. , Lotzova, E. and Pluznik, D.H. (Eds.), Experimental Haematology today - 1988. Springer Verlag, New York:pp. 92-99 (1989)
• baculoviruses Luckow et al; Bio-technology 6: 47-55 (1988) .
• the invention also comprises a virus vector containing a nucleic acid sequence encoding the polypeptide fragment, or a recombinant nucleic acid molecule encoding the polypeptide fragment under the control of regulating sequences enabling expression of the protein encoded by said nucleic acid sequence.
• the invention comprises a host cell containing a nucleic acid sequence encoding the polypeptide fragment, or a recombinant nucleic acid molecule encoding the polypeptide fragment under the control of regulating sequences enabling expression of the protein encoded by said nucleic acid sequence.
• the invention also comprises a host cell containing a virus vector containing a nucleic acid molecule encoding the polypeptide fragment, or a recombinant nucleic acid molecule encoding the polypeptide fragment under the control of regulating sequences enabling expression of the protein encoded by said nucleic acid sequence.
• a host cell may be a cell of bacterial origin, e.g. Escherichia coli. Bacillus subtilus and Lactobacillus species, in combination with bacteria- based vectors as pBR322, or bacterial expression vectors as pGEX, or with bacteriophages.
• the host cell may also be of eukaryotic origin, e.g. yeast-cells in combination with yeast-specific vector molecules, or higher eukaryotic cells like insect cells (Luckow et al; Bio-technology 6: 47-55 (1988)) in combination with vectors or recombinant baculoviruses, plant cells in combination with e.g. Ti-plasmid based vectors or plant viral vectors (Barton, K.A. et al; Cell 32: 1033 (1983) , mammalian cells like Hela cells, Chinese Hamster Ovary cells (CHO) or Crandell Feline Kidney- cells, also with appropriate vectors or recombinant viruses.
• a vaccine for the protection of cats against Feline Immuno-deficiency Virus infections can be made.
• the vaccine may comprise said nucleic acid sequence or a recombinant nucleic acid molecule as explained above or said vector virus or said host cell.
• the vaccine may also comprise the polypeptide fragment mentioned before or the immunogen mentioned above.
• the vaccine also comprises an adjuvant.
• adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner.
• a number of different adjuvants are known in the art. Examples of adjuvants are Freunds Complete and Incomplete adjuvant, vitamin E, non-ionic block polymers, muramyldipeptides, Quill AW, mineral oil e.g. Bayol (R) or Markol ⁇ , vegetable oil, and Carbopol( R ) (a homopolymer).
• the vaccine may also comprise a so-called "vehicle".
• a vehicle is a compound, or to which the polypeptide adheres, without being covalently bound to it.
• vehicle compounds are e.g. aluminium hydroxide, -phosphate or -oxide, silica, Kaolin, and Bentonite
• the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.
• .Another method for obtaining antibodies is the method for making so-called monoclonal antibodies. It depends on the production and selection of one specific antibody type reactive with one specific epitope.
• the method for production of monoclonal antibodies by using the hybridoma technique has been published a.o. by Kohler and Milstein (Nature 256: 459 (1975)), Kohler and Milstein (Eur. J. Immunol. 6: 511 (1976)), Gefter et al (Somatic Cell Genet. 3: 231 (1977)), Volk et al (J. Virol. 42: 220 (1982)) and Hammerling et al (Monoclonal Antibodies and T-Cell Hybridomas, Elsevier New York, pp. 563-681 (1981)).
• mouse monoclonal antibodies were made that were shown to be reactive with an epitope, located on the Central Fragment of the FIV surface protein.
• mice were vaccinated twice with sufficiently large doses of an inactivated whole virus preparation, in order to obtain a clear anti-FIV antibody response. Fusions were made after antibody response was reached, between myeloma cells and mouse spleen cells. Hybridomas were tested for antibody production, and among positive clones, i.a. a hybridoma was found to produce a neutralising epitope recognising a conformational epitope of the FIV surface protein located in the Central Fragment.
• the invention thus relates to monoclonal antibodies that are reactive with the polypeptide as described in SEQ ID NO: 4 or a portion thereof, or immunologically active variants thereof.
• the monoclonal antibody is from the hybridoma 1E1EB4-93030567 deposited with the ECACC.
• the present invention also relates to the use of the polypeptide or the immunogen for the preparation of a vaccine for the prophylaxis of Feline Immuno- deficiency Virus infection.
• Genomic DNA of FIV-113 infected cells was isolated and digested to completion with Nhel. Fragments hybridizing with both the Pol-gene and the U3-R region of the FIV-LTR, and thus comprising the genetic information for the surface protein, were used for further subcloning and subsequently sequenced. All DNA-techniques were carried out essentially as described by Sambrook (Sambrook et al. Molecular cloning, a laboratory manual. Cold Spring Laboratory Press, Cold Spring Harbor, New York (1989)).
• the sequence comprising the FIV-surface protein code is given in SEQ ID NO: 1.
• the pOTSKF33 plasmid vector (Chiang et al, Clin. Chem. 35: 946-952 (1989), Krone et al, J. Med. Virol 26: 261-270 (1988)) encoding the amino-terminal part of galactokinase (galK) controlled by an inducible promoter was used to construct fusion proteins between galactokinase and the surface protein of FIV strain UT113. Standard cloning techniques (Sambrook et al. Molecular cloning, a laboratory manual.
• the galK-CT fusion encoding the carboxyl-part of the surface protein (further referred to as CT) spanning amino acids 516 - 611 was constructed using PCR with the carboxyl end at the cleavage site between the surface and transmerobrane (TM) protein.
• the galK-CT5T fusion is identical to galK-CT with a deletion from amino acid 599 to 611.
• the galK-CF, galK-CT and galk-CTffT fusion proteins were used to develop an ELISA for the detection of surface specific antibodies in sera of FIV-infected cats.
• Sera of cats prior to infection with FIV did not show FIV CF, CT and CT ⁇ * T specific antibodies indicating the specificity of the ELISA (table 1) .
• All cats showed a seroconversion for antibodies to at least one of the CF and CT proteins. The seroconversion occurred starting from week 6 after FIV infection depending on the isolate and dose of inoculation used. All cats showed antibodies to CF, albeit that the levels of antibodies showed some variation.
• the second best recognized protein was CT against which in 15 out of 24 cat sera antibodies could be detected.
• Rabbits (New Zealand white) were injected subcutaneously with 100 ⁇ g of the galK-CF or galk-CT fusion protein in Freunds complete adjuvant. Every three weeks the rabbits were boosted with 100 ⁇ g of the galK-CF or galk-CT fusion protein in Freunds incomplete adjuvant. Hyperimmune sera reacted on immuno-blots with the FIV surface protein as produced in CRFK cells and in a baculo virus based expression system.
• Outbred cats were injected subcutaneously with 100 ⁇ g of the galK-CF protein in an oil/alum adjuvant supplemented with G-MDP. Every six weeks the cats received a booster injection. Hyperimmune sera reacted with the FIV surface protein as produced in a baculo virus based expression system.
• CRFK cells (Crandell et al, In vitro 9: 176-185 (1973)) (3500/well) were seeded in an 96-well plate and maintained in DMEM supplemented with 5% fetal calf serum.
• 50 TCID50 of CRFK derived FIV-UT113 was incubated for 1 hour at 37 *C with serial dilutions of the serum to be assayed.
• CRFK cells were washed with PBS + DEAE (50 ⁇ g/ml) and were incubated with the virus/serum mixture.
• CRFK cells were washed with PBS and subsequently propagated in DMEM supplemented with 2% fetal calf serum.
• the supernatant of the CRFK cells was assayed for viral p24 gag production. An inhibition of p24 production greater than 90% was considered as neutralization.
• the neutralizing rabbit serum, a neutralizing monoclonal, a representative neutralizing cat serum, and control rabbit and cat sera were analysed with overlapping short polypeptides, together representing the whole surface amino acid sequence contained within the CF fusion protein.
• Both the cat serum and the rabbit serum recognized peptides with the core sequence WRPDFE (amino acids 402-407) .
• the cat serum recognized a wider spectrum of peptides including the WRPDFE core sequence and apparently consisting of multiple core sequences encompassing the SWKQGNRWEWRPDFESERV stretch of amino acids (amino acids 393-411) . Results of the scanning are given in figure 2.
• the neutralising monoclonal antibody does not directly react with the CF protein, but is prevented from binding to the FIV surface protein by the polyclonal rabbit serum against the Central Fragment polypeptide, synthesized in bacteria. This indicates that a similar region of the surface protein is recognised by rabbit as well as mouse antibodies. It is also concluded, that the mouse monoclonal antibody is directed to a conformational epitope.
• the CF region of the surface protein of FIV contains a neutralising domain of linear as well as conformational architecture capable of eliciting neutralising antibodies against FIV in cats.
• Sera of cats infected with different FIV isolates were screened by ELISA for antibodies against the surface protein fragments CF, CT and CT ⁇ * T.
• Sera were tested in a neutralization assay.
• the reciprocal neutralization titers of pre-immune rabbit and cat sera were less than 10.
• Hyper-immune rabbit sera were derived from rabbits which received at least two booster injections. The cats received one booster injection.
• the pool of sera of FIV infected cats was derived from cats infected with FIV-Ktj (cats 320, 322, 326), and a reciprocal neutralizing titer which was relatively high amongst FIV infected cats tested so far.
• Envelope surface fragments were constructed as described (materials and methods) using convenient restriction enzyme sites and primers for PCR.
• Overlapping 12-mer peptides of CF (FIV surface protein amino acids 361-372, 362-373, etc. to 433-445) were synthesized on a solid support and serum antibodies were detected using ELISA.
• MOLECULE TYPE RNA (genomic)
• ORGANISM Feline immunodeficiency virus
• GCT CTA AGG AAT GAA ATT CAA GAG GTA AAA CTG GAA GAA GGA AAT GCA 240 Ala Leu Arg Asn Glu He Gin Glu Val Lys Leu Glu Glu Gly Asn Ala 65 70 75 80
• AGA AAA AGG TTT GGG TCC TTA
• MOLECULE TYPE RNA (genomic)
• ORGANISM Feline immunodeficiency virus
• TGT CAA AGA ACA CAG AGT CAG CCT GGG TCA TGG ATT AGG GCA ATC TCG 96 Cys Gin Arg Thr Gin Ser Gin Pro Gly Ser Trp He Arg Ala He Ser 20 25 30

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