CN102775478A - Foot and mouth disease virus antigen peptide and vaccine - Google Patents

Foot and mouth disease virus antigen peptide and vaccine Download PDF

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Publication number
CN102775478A
CN102775478A CN2012103010383A CN201210301038A CN102775478A CN 102775478 A CN102775478 A CN 102775478A CN 2012103010383 A CN2012103010383 A CN 2012103010383A CN 201210301038 A CN201210301038 A CN 201210301038A CN 102775478 A CN102775478 A CN 102775478A
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foot
thr
gly
mouth disease
antigen polypeptide
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CN102775478B (en
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陈智英
郑海学
刘湘涛
才学鹏
聂东升
殷宏
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Shen Lian biological medicine (Shanghai) Limited by Share Ltd
Lanzhou Veterinary Research Institute of CAAS
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Shen Lian Biotechnology Co ltd
Lanzhou Veterinary Research Institute of CAAS
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Abstract

The invention provides a foot and mouth disease virus antigen peptide, a fusion antigen peptide and a foot and mouth disease virus vaccine containing the antigen peptide and/or the fusion antigen peptide. The invention also provides a preparation method of the antigen peptide, the fusion antigen peptide and the vaccine. The invention further provides applications of the antigen peptide, the fusion antigen peptide and the vaccine in the aspect of preventing and controlling virus infection of foot and mouth diseases. The foot and mouth disease virus antigen peptide, the fusion antigen peptide and the vaccine have broad-spectrum immunogenicity and can have good immunogenicity on different foot and mouth disease viruses and variants thereof.

Description

Foot-and-mouth disease virus antigen polypeptide and vaccine
Technical field
The present invention relates to the foot-and-mouth disease virus antigen polypeptide, fused antigen polypeptide, and the foot and mouth disease virus vaccine that contains above-mentioned antigenic peptide and/or fused antigen polypeptide.The invention still further relates to above-mentioned antigenic peptide, the preparation of fused antigen polypeptide and vaccine and application thereof.
Background technology
Foot and mouth disease be by foot and mouth disease virus caused a kind of acute, hot, the height contagious infection zoonosis.Foot and mouth disease is mainly encroached on cloven-hoofed animal, and its clinical diagnosis is characterized as oral mucosa, hoof and skin of breast generation blister.It is high but its sickness rate can reach 100% that adult animals infects after the foot and mouth disease general lethality rate, and a large amount of blisters can appear in mouth, the hoof of morbidity animal, and are spiritual depressed, and then make animal yield fall sharply.Cub then loses the sudden death of symptom ground through regular meeting after infecting foot and mouth disease, and lethality rate can reach 100% when serious.Because foot and mouth disease is propagated rapidly, is difficult to control, remedial measures is few, can only butcher with collective after therefore each outburst and destroy the livestock that catches an illness by fire with trouble without offspring, thereby cause enormous economic loss for the herding industry.
Foot and mouth disease virus is the pathogenic former of foot and mouth disease, and it belongs to picornavirus, has characteristics such as many serotypes, high variability.Known foot and mouth disease virus has O, A, C, SAT1 (South Africa I type), SAT2 (South Africa II type), SAT3 (South Africa III type) and 7 main serotypes of Asia1 (Asia I type) at present.Every kind of principal mode divides some hypotypes again, the existing kind more than 70 of the hypotype of finding at present.Antigenicity between foot and mouth disease virus is various is usually different; Immunity alternately each other; And the degree of antigenic variation is also very big in a kind of serotype, maybe be to but not protection of the another kind of hypotype in the same serotype to such an extent as to can resist a kind of aftosa vaccine of hypotype effectively.
Because immunity each other usually between the high mutation property of foot and mouth disease virus and each serotype is so bring huge difficulty for the preventing and controlling of foot and mouth disease.Still press at present to provide foot and mouth disease virus is had good immunogenic vaccine, the foot and mouth disease virus that the ideal vaccine is also tackled multiple different subtype all has good immunogenicity, thereby can immunoprotection widely be provided for livestock.
Summary of the invention
The present invention relates to the foot-and-mouth disease virus antigen polypeptide; The fused antigen polypeptide; And the foot and mouth disease virus vaccine that contains above-mentioned antigenic peptide and/or fused antigen polypeptide; This antigenic peptide can make host animal produce the immunogenicity to multiple different pedigree foot and mouth disease viruses with vaccine, thereby for host animal the immunoprotection that is directed against multiple different pedigree foot and mouth disease viruses is provided.
With regard to the present invention; Term " immunity " is meant; With suitable form and through suitable approach to host animal give with a certain antigen after, it is antigenic specific cell-mediated and/or based on the immunne response of antibody, for example to induce host animal to produce to this; The activation of antibody and/or production of cytokines and/or cytotoxic T cell, antigen presenting cell, helper cell, dendritic cell, and/or other cell response reaction.
With regard to the present invention, term " immunogenicity " is meant that one or more antigen induction host animals produce to this one or more antigenic specific immunoreactive ability.
With regard to the present invention, term " antigenic peptide " be meant by two or more amino acid through peptide bond covalently bound form have an immunogenic peptide chain structure.Peptide chain structure of the present invention can be a straight chain, the band branched structure, and/or band shape structure.
With regard to the present invention, term " vaccine " be meant contain that one or more have the antigen of body immunity and be formulated into can be to the preparation of host animal form of medication.After being incorporated into vaccine in the host animal body, this vaccine just can excite host animal to produce to this one or more antigenic immunoreation.
With regard to the present invention, term " host animal " is meant can be by the animal that foot and mouth disease virus is invaded and the permission foot and mouth disease virus is duplicated in its body.In some embodiments, host animal is an artiodactyl.In some embodiments, host animal is livestock animals such as pig, ox, sheep.The intrusion and duplicate of foot and mouth disease virus in host animal possibly make host animal produce the Clinical symptoms of foot and mouth disease, also possibly not make host animal produce any Clinical symptoms.
The invention provides the foot-and-mouth disease virus antigen polypeptide of one type of synthetic.According to an aspect of the present invention, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val Leu X 8X 9Lys Ala Ala ArgX 10Leu Pro Thr Ser Phe Asn Tyr X 11Ala Ile Lys (SEQ ID NO:1), wherein, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro, X 10=Thr or Cys, X 11=Gly or Cys, and X 10And X 11Must there be one and can only have one to get Cys.
In one embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val Leu X 8X 9Lys Ala Ala ArgCys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:2), wherein, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val Leu X 8X 9Lys Ala Ala ArgThr Leu Pro Thr Ser Phe Asn Tyr Cys Ala Ile Lys (SEQ ID NO:3), wherein, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Cys?Lys?Tyr?Gly?Glu?Ser?Pro?Val?Ala?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Thr?Pro?Lys?AlaAla?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：4）。
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Cys?Lys?Tyr?Gly?Glu?Ser?Pro?Val?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Thr?Gln?Lys?AlaAla?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：5）
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Cys?Lys?Tyr?Gly?Asp?Gly?Thr?Val?Ala?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?AlaAla?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：6）。
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Cys?Lys?Tyr?Gly?Glu?Ser?His?Thr?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?LysAla?Ala?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：7）。
According to another aspect of the present invention, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly X 1Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val Leu X 8X 9Lys Ala Ala Arg X 10Leu Pro Thr Ser Phe Asn Tyr X 11Ala Ile Lys (SEQ ID NO:8), wherein, X 1=Asn or Asp, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro, X 10=Thr or Cys, X 11=Gly or Cys, and X 10And X 11Must there be one and can only have one to get Cys.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly X 1Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val Leu X 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:9), wherein, X 1=Asn or Asp, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly X 1Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val Leu X 8X 9Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Cys Ala Ile Lys (SEQ ID NO:10), wherein, X 1=Asn or Asp, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val LeuX 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:11), wherein, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val LeuX 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr GlyAla Ile Lys (SEQ ID NO:12), wherein, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val LeuX 8X 9Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr CysAla Ile Lys (SEQ ID NO:13), wherein, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val LeuX 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ IDNO:14), wherein, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
According to another aspect of the present invention, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val LeuX 8X 9Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Cys Ala Ile Lys (SEQ ID NO:15), wherein, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro X 6X 7Asn Val Arg Gly Asp Leu Gln ValLeu X 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:16), wherein, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro X 6X 7Asn Val Arg Gly Asp Leu Gln ValLeu X 8X 9Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Cys Ala Ile Lys (SEQ ID NO:17), wherein, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val X 7Asn Val Arg Gly Asp Leu Gln ValLeu X 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:18), wherein, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val X 7Asn Val Arg Gly Asp Leu Gln ValLeu X 8X 9Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Cys Ala Ile Lys (SEQ ID NO:19), wherein, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val Ala Asn Val Arg Gly Asp Leu Gln ValLeu X 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:20), wherein, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val Ala Asn Val Arg Gly Asp Leu Gln ValLeu X 8X 9Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Cys Ala Ile Lys (SEQ ID NO:21), wherein, X 8=Thr or Ala, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val Ala Asn Val Arg Gly Asp Leu Gln ValLeu Thr X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr GlyAla Ile Lys (SEQ ID NO:22), wherein, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val Tyr Asn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val Ala Asn Val Arg Gly Asp Leu Gln ValLeu Thr X 9Lys Ala Ala Arg Thr Leu Pro Thr Ser Phe Asn Tyr Cys Ala Ile Lys (SEQ ID NO:23), wherein, X 9=Gln or Pro.
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Ser?Pro?Val?Ala?Asn?Val?Arg?Gly?Asp?Leu?Gln?ValLeu?Thr?Pro?Lys?Ala?Ala?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：24）。
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Ser?Pro?Val?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?ValLeu?Thr?Gln?Lys?Ala?Ala?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：25）。
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Asp?Gly?Thr?Val?Ala?Asn?Val?Arg?Gly?Asp?Leu?Gln?ValLeu?Ala?Gln?Lys?Ala?Ala?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：26）。
In another embodiment, antigenic peptide provided by the invention contains aminoacid sequence as follows:
Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Ser?His?Thr?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?ValLeu?Ala?Gln?Lys?Ala?Ala?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：27）。
Antigenic peptide of the present invention comprises above-mentioned aminoacid sequence and carries out the sequence that one or more amino acid whose replacement, interpolation and/or deletion produced at one or more amino acid sites.The amino acid replacement comprises conservative replacement and non-conservative replacement.Conservative replacement is meant the replacement between the amino acid of similar performance; The for example replacement between the polare Aminosaeren (like the replacement between Stimulina and the l-asparagine); Replacement between the hydrophobic amino acid (like the replacement between leucine, Isoleucine, methionine(Met) and the Xie Ansuan); And the replacement between the band amino acids with identical charges (like the replacement between l-arginine, Methionin and the Histidine, the perhaps replacement between L-glutamic acid and the aspartic acid) etc.Non-conservative replacement is meant the replacement between the amino acid of different nature, and the non-conservative replacement that the present invention comprised will can not change specific immunity originality and the activity of said antigenic peptide to foot and mouth disease virus.Amino acid whose interpolation of the present invention and/or deletion will be no more than 1, and 2,3,4,5,6,7,8,9 or 10 amino acid.According to an aspect of the present invention, be no more than 1 in the N of above-mentioned aminoacid sequence (the for example sequence of SEQ ID NOs:1-27) end interpolation and/or deletion, 2,3,4,5,6,7,8,9 or 10 amino acid; According to another aspect of the present invention, be no more than 1 in the C of above-mentioned aminoacid sequence (the for example sequence of SEQ IDNOs:1-27) end interpolation and/or deletion, 2,3,4,5,6,7,8,9 or 10 amino acid; According to another aspect of the present invention, hold interpolation and/or deletion to be no more than 1 at the N of above-mentioned aminoacid sequence (the for example sequence of SEQ IDNOs:1-27) end and/or C, 2,3,4,5,6,7,8,9 or 10 amino acid.
According to another aspect of the present invention, the invention provides one type of fused antigen polypeptide that comprises two or more antigenic peptides of the present invention.A plurality of antigenic peptides that the fused antigen polypeptide is comprised covalently or non-covalently connect each other.Covalently bound for example can be that perhaps C interconnects through ester bond, ehter bond, phosphoric acid ester bond, amido linkage, peptide bond, imide key, carbon-sulfide linkage.Non-covalent connection for example can be that perhaps hydrogen bond interconnects through interionic interaction force, hydrophobic interaction.
In an embodiment of the invention, the contained a plurality of antigenic peptides of fused antigen polypeptide connect through connection chain.With regard to the present invention, term " connection chain " is meant, is used for covalently or non-covalently connecting the structural unit of two adjacent peptide sequences, and the introducing of this structural unit can not produce infringement to the immunogenicity of fused antigen polypeptide.Connection chain can comprise one or more amino-acid residues, and connection chain can also comprise for example C 1-C 6Alkyl, C 3-C 6Structures such as naphthenic base, aryl or heteroaryl.Prior art has disclosed some known connection chains, and those skilled in the art can easily select as required.
In yet another embodiment of the present invention, connection chain used in the fused antigen polypeptide is by one or more Methionins, L-Ala, and glycocoll, Serine and/or proline(Pro) or its arbitrary combination are formed.For example connection chain is by 1, and 2,3,4,5,6,7,8,9,10 or more a plurality of aforementioned amino acid or its arbitrary combination are formed.In yet another embodiment of the present invention; Used connection chain is the small peptide of being made up of one or more Methionins in the fused antigen polypeptide, short peptide sequence Lys for example, Lys Lys; Lys Lys Lys; Lys Lys Lys Lys, Lys Lys Lys Lys Lys, Lys Lys LysLys Lys Lys.In yet another embodiment of the present invention, the used connection chain of being made up of one or more Methionins comprises one or more ε-Methionins in the fused antigen polypeptide.In yet another embodiment of the present invention, used connection chain is to mix the small peptide of forming, for example Lys Ala Lys by one or more Methionins and L-Ala in the fused antigen polypeptide; Ala Lys Ala; Lys Ala Ala Lys, Ala Lys Lys Ala, Lys Lys Ala Lys; Ala Lys Ala Lys, Lys Lys Ala Lys Lys or Lys Ala Lys Lys Ala Lys.In yet another embodiment of the present invention, used connection chain is by one or more proline(Pro) in the fused antigen polypeptide, and/or the polypeptide of Serine and/or glycocoll mixing composition, for example Pro Ser Pro; Ser ProSer, Ser Ser Pro, Pro Pro Ser, Gly Ser Gly; Ser Gly Ser, Ser Ser Gly Ser, Gly Gly Ser Gly; Gly Ser Gly Ser, Gly Ser Gly Ser Ser, Gly Gly Gly Gly Ser.
The antigenic peptide quantity that the fused antigen polypeptide is comprised for example can be 2,3, and 4,5,6,7,8,9,10 or more a plurality of.The antigenic peptide that is comprised in the fused antigen polypeptide can be identical also can be inequality.For example, the fused antigen polypeptide comprises two antigenic peptides shown in the SEQ IDNO.1, perhaps comprises two antigenic peptides shown in the SEQ IDNO.2, also or comprise the antigenic peptide shown in an antigenic peptide shown in the SEQ ID NO.1 and the SEQ ID NO.2.
The antigenic peptide that the fused antigen polypeptide comprises can be natural foot-and-mouth disease virus antigen peptide sequence, also can be the foot-and-mouth disease virus antigen polypeptide of synthetic.In an embodiment of the invention, the fused antigen polypeptide comprises one or more antigenic peptide sequences provided by the invention and one or more natural antigen peptide sequence.For example, the fused antigen polypeptide comprises one section antigenic peptide sequence provided by the invention and one section natural antigen peptide sequence.Again for example, the fused antigen polypeptide comprises at least one section antigenic peptide sequence provided by the invention and at least one section natural antigenic peptide sequence.
In yet another embodiment of the present invention, fused antigen polypeptide of the present invention comprises all or part of aminoacid sequence of foot and mouth disease virus polyprotein.A kind of natural acid sequence of known O type foot and mouth disease virus polyprotein contains 104 amino acid, and its aminoacid sequence is following:
His?Arg?Val?Leu?Ala?Thr?Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Ser?His?Thr?Thr?Asn?ValArg?Gly?Asp?Leu?Gln?Val?Leu?Ala?Gln?Lys?Ala?Ala?Arg?Thr?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?AlaIle?Lys?Ala?Thr?Arg?Val?Thr?Glu?Leu?Leu?Tyr?Arg?Met?Lys?Arg?Ala?Glu?Thr?Tyr?Cys?Pro?Arg?ProLeu?Leu?Ala?Ile?His?Pro?Asn?Glu?Ala?Arg?His?Lys?Gln?Lys?Ile?Val?Ala?Pro?Val?Lys?Gln?Leu?LeuAsn?Phe?Asp?Leu?Leu?Lys?Leu?Ala?Gly?Asp?Val?Glu?Ser（GenBank?No.:ABU87566.1）（SEQ?ID?NO：28）。
In another aspect of the present invention, the invention provides one type of fused antigen polypeptide that comprises antigenic peptide of the present invention and t helper cell epi-position.Antigenic peptide that the fused antigen polypeptide is comprised and t helper cell epi-position can be respectively one or more, and for example the fused antigen polypeptide comprises 1 respectively, 2, and 3; 4,5,6,7; 8,9,10 or more a plurality of antigenic peptide and t helper cell epi-position.The antigenic peptide that the fused antigen polypeptide is comprised can be identical also can be inequality, the t helper cell epi-position that is comprised can be identical also can be inequality.Can connect through himself amino acids formed peptide bond between adjacent antigenic peptide and the t helper cell epi-position, between the adjacent antigenic peptide or between the adjacent t helper cell epi-position; Also or other any-mode of mentioning through the aforementioned part of the present invention be connected, for example connect antigenic peptide and t helper cell epi-position through connection chain.
The t helper cell epi-position of here addressing is meant the aminoacid sequence that supplies t helper cell identification on the antigen.T helper cell is meant one type of specific T cell, thereby its effect is auxiliary T killer cell to be made the T killer cell combine with target antigen or cell and eliminate or kill this target antigen or cell.Prior art has disclosed some known t helper cell epi-positions, and those skilled in the art can select suitable t helper cell epi-position and are used in the fused antigen polypeptide of the present invention on understanding the basis of this purpose according to the technological general knowledge of its grasp.The instance of t helper cell epi-position includes but not limited to; Hepatitis B virus surface and cAg t helper cell epi-position, Toxins, pertussis t helper cell epi-position, tetanus toxin t helper cell epi-position, Measles virus F albumen t helper cell epi-position, chlamydia trachomatis major outer membrane albumen t helper cell epi-position, diphtheria toxin t helper cell epi-position, plasmodium falciparum ring sporophyte t helper cell epi-position, graceful Sen Shi fluke trisaccharide phosphoric acid salt isomerase t helper cell epi-position, and intestinal bacteria TraT t helper cell epi-position.
In an embodiment of the invention, the t helper cell epi-position is the t helper cell epi-position based on Measles virus, and it comprises aminoacid sequence as follows:
Leu?Ser?Glu?Ile?Lys?Gly?Val?Ile?Val?His?Arg?Leu?Glu?Gly?Val（SEQ?ID?NO:29）。
In yet another embodiment of the present invention, the t helper cell epi-position is the t helper cell epi-position based on Measles virus, and it comprises aminoacid sequence as follows:
Gly Ile Leu Glu X 1X 2Gly Ile X 3Ala X 4Ile X 5X 6Thr Glu Leu Ile Phe (SEQ ID NO:30), wherein X 1=Ser or Thr, X 2=Arg or Lys, X 3=Lys or Arg, X 4=Arg or Lys, X 5=Thr or Gly, X 6=His or Arg.
In yet another embodiment of the present invention, the t helper cell epi-position is the t helper cell epi-position based on Measles virus, and it comprises aminoacid sequence as follows:
Gly?Ile?Leu?Glu?Ser?Arg?Gly?Ile?Lys?Ala?Arg?Ile?Thr?His?Thr?Glu?Leu?Ile?Phe（SEQ?ID?NO：31）。
In yet another embodiment of the present invention, the t helper cell epi-position is the t helper cell epi-position based on Measles virus, and it comprises aminoacid sequence as follows:
Gly?Ile?Leu?Glu?Thr?Arg?Gly?Ile?Lys?Ala?Arg?Ile?Thr?His?Thr?Glu?Leu?Ile?Phe（SEQ?ID?NO：32）。
In yet another embodiment of the present invention, the t helper cell epi-position is the t helper cell epi-position based on Measles virus, and it comprises aminoacid sequence as follows:
Gly?Ile?Leu?Glu?Thr?Lys?Gly?Ile?Lys?Ala?Arg?Ile?Thr?His?Thr?Glu?Leu?Ile?Phe（SEQ?ID?NO：33）。
In yet another embodiment of the present invention, the t helper cell epi-position is the t helper cell epi-position based on Measles virus, and it comprises aminoacid sequence as follows:
Gly?Ile?Leu?Glu?Thr?Lys?Gly?Ile?Arg?Ala?Lys?Ile?Gly?Arg?Thr?Glu?Leu?Ile?Phe（SEQ?ID?NO：34）。
The non-limiting example that includes the fused antigen polypeptide of antigenic peptide and t helper cell epi-position for example is:
Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val ε-Lys Val Tyr Asn Gly Asn CysLys Tyr Gly Glu Ser Pro Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu Thr Pro Lys Ala Ala ArgCys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:35); Or
Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val ε-Lys Val Tyr Asn Gly Asn CysLys Tyr Gly Glu Ser Pro Val Thr Asn Val Arg Gly Asp Leu Gln Val Leu Thr Gln Lys Ala Ala ArgCys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:36); Or
Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val ε-Lys Val Tyr Asn Gly Asn CysLys Tyr Gly Asp Gly Thr Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala ArgCys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:37); Or
Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val ε-Lys Val Tyr Asn Gly Asn CysLys Tyr Gly Glu Ser His Thr Thr Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala ArgCys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:38), or
Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr His T hr Glu Leu Ile Phe ε-Lys Val TyrAsn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu Thr ProLys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:39); Or
Gly Ile Leu Glu Thr Arg Gly Ile Lys Ala Arg Ile Thr His Thr Glu Leu Ile Phe ε-Lys Val TyrAsn Gly Asn Cys Lys Tyr Gly Glu Ser Pro Val Thr Asn Val Arg Gly Asp Leu Gln Val Leu Thr GlnLys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:40); Or
Gly Ile Leu Glu Thr Lys Gly Ile Lys Ala Arg Ile Thr His Thr Glu Leu Ile Phe ε-Lys Val TyrAsn Gly Asn Cys Lys Tyr Gly Asp Gly Thr Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu AlaGln Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:41); Or
Gly?Ile?Leu?Glu?Thr?Lys?Gly?Ile?Arg?Ala?Lys?Ile?Gly?Arg?Thr?Glu?Leu?Ile?Phe?ε-Lys?Val?TyrAsn?Gly?Asn?Cys?Lys?Tyr?Gly?Glu?Ser?His?Thr?Thr?Asn?Val?Arg?Gly?Asp?Leu?Gln?Val?Leu?Ala?GlnLys?Ala?Ala?Arg?Cys?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys（SEQ?ID?NO：42）。
USP U.S.7; 744; No. 898 (applying date is on September 8th, 2005; Denomination of invention is " Peptides for Inducing Cytotoxic T Lymphocyte Responses to Hepatitis B Virus "), and USP U.S.7,833; No. 532 (its applying date is on August 12nd, 2003, denomination of invention for " Immunogenic Lipopeptides Comprising T-helper and Cytotoxic T Lymphocyte (CTL) Epitopes) in introduced multiple t helper cell epi-position.For example, U.S.7,744,898 specification sheetss have listed the wherein t helper cell epi-position shown in the SEQ ID NOs.6-9.Again for example, U.S.7 has listed wherein SEQ IDNOs.1 in 833,532 specification sheetss, the described t helper cell epi-position of 14-52.U.S.7,744,898 and U.S.7,833,532 this by reference mode be incorporated in full among the application with for referencial use.
In another aspect of the present invention, the invention provides one type of fused antigen polypeptide that comprises antigenic peptide of the present invention and immunostimulatory sequence.Antigenic peptide that the fused antigen polypeptide is comprised and immunostimulatory sequence can be respectively one or more, and for example the fused antigen polypeptide comprises 1 respectively, 2, and 3,4,5,6,7,8,9,10 or more a plurality of antigenic peptide and immunostimulatory sequence.The antigenic peptide that the fused antigen polypeptide is comprised can be identical also can be inequality, the immunostimulatory sequence that is comprised can be identical also can be inequality.Can connect through himself amino acids formed peptide bond between the adjacent antigenic peptide and immunostimulatory sequence, between the adjacent antigenic peptide or between the adjacent immunostimulatory sequence; Also or other any-mode of mentioning through the aforementioned part of the present invention be connected, for example connect antigenic peptide and immunostimulatory sequence through connection chain.
The immunostimulatory sequence of here addressing be meant can the enhancement antigen immunogenicity of polypeptides material; The structural domain of the invasin albumen (Inv) that for example belongs to from the bacterium yersinia (people such as Brett; The Europe Journal of Immunology; 1993,23:1608-1614) or from the immunostimulation analogue of respective regions in other yersinia invasin albumen.Immunostimulatory sequence commonly used is the known technology of this area, and those skilled in the art can select suitable immunostimulatory sequence and are used in the fused antigen polypeptide of the present invention according to the technological general knowledge of its grasp.
In another aspect of the present invention, the invention provides one comprise antigenic peptide of the present invention, t helper cell epi-position and immunostimulatory sequence roughly the same the time the fused antigen polypeptide.Antigenic peptide, t helper cell epi-position and the quantity of immunostimulatory sequence in the fused antigen polypeptide can be respectively one or more, for example 1, and 2,3,4,5,6,7,8,9,10 or more a plurality of.For example, the fused antigen polypeptide comprises an antigenic peptide, two or more t helper cell epi-positions, and two or more immunostimulatory sequences.Again for example, the fused antigen polypeptide comprises two or more antigenic peptides, a t helper cell epi-position, and an immunostimulatory sequence.Again for example, the fused antigen polypeptide comprises two or more antigenic peptides, two or more t helper cell epi-positions, and two or more immunostimulatory sequences.Can be connected through the any-mode that the aforementioned part of the present invention is mentioned between adjacent antigenic peptide, t helper cell epi-position and the immunostimulatory sequence, for example connect, for example connect again through the peptide bond that forms between himself amino acid through connection chain.Be to be understood that here; The antigenic peptide that is comprised in the fused antigen polypeptide, t helper cell epi-position and immunostimulatory sequence can be arranged with any-mode; For example antigenic peptide directly is connected with immunostimulatory sequence with the t helper cell epi-position respectively; For example the t helper cell epi-position directly is connected with immunostimulatory sequence with antigenic peptide respectively again, and for example immunostimulatory sequence directly is connected with the t helper cell epi-position with antigenic peptide respectively again.
In another aspect of the present invention, the peptide chain of antigenic peptide provided by the invention or fused antigen polypeptide can have ring texture.Said ring texture can ways of connecting forms through between two or more amino acid of peptide chain, forming covalently or non-covalently, for example between two or more amino acid, forms disulfide linkage, peptide bond; Carbon bond (for example C-C or C=C); Ester bond, ehter bond, azo bond; The C-S-C key, the mode of C-N-C key or C=N-C key forms.In an embodiment of the invention, antigenic peptide or fused antigen polypeptide form ring texture through between two halfcystines of himself, forming disulfide linkage.In an embodiment of the invention, the antigenic peptide that comprises sequence shown in the SEQ ID NOs:1-27 has ring texture.In an embodiment of the invention, comprising the ring texture that the antigenic peptide of sequence shown in the SEQ ID NOs:1-27 has forms by forming disulfide linkage between halfcystine.
The polypeptide cyclisation can realize through the known technology of this area, for example can come that polypeptide is carried out cyclisation through natural method and handle, and handles the acquisition cyclized structure thereby perhaps come that through chemical synthesis process polypeptide is carried out cyclisation.Document " people such as Anwer; Int.J Pep.Protein Res.36:392-399 (1990) " and " people such as Rivera-Baeza; Neuropeptides30:327-333 (1996) " write up the method for polypeptide cyclisation, they this by reference mode incorporate the application in full into for referencial use.
In an embodiment of the invention, the invention provides a kind of cyclisation degree greater than 85% antigenic peptide and fused antigen polypeptide.In an embodiment of the invention, the invention provides a kind of cyclisation degree and be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% antigenic peptide and fused antigen polypeptide.In yet another embodiment of the present invention, the invention provides a kind of cyclisation degree greater than 87.5% antigenic peptide and fused antigen polypeptide." cyclisation degree " used herein is meant that the peptide chain that has ring texture in antigenic peptide or the fused antigen polypeptide accounts for the per-cent of total peptide chain number.Those skilled in the art can select suitable method to measure the cyclisation degree according to the form of the chemical bond that forms ring texture.For example, if form ring texture through disulfide linkage, its cyclisation degree can be measured through the Ellman method of standard, and this cyclisation degree is calculated as the per-cent that accounts for initial total free sulfhydryl group (SH) number through sulfydryl (SH) number of disulfide linkage Cheng Huan.The Ellman method belongs to the known technology of this area, its write up in document " people such as Ellman, Anal.Biochem.94:75-81 (1979) ", the document this by reference mode incorporate the application in full into for referencial use.Simply; The Ellman method can be measured the content of free sulfhydryl group in the polypeptide (SH), uses the Ellman method to measure polypeptide cyclisation degree and may be summarized to be following key step: prepare the halfcystine solution of a plurality of different concns and add Ellman reagent respectively and react; The absorbancy of free sulfhydryl group (SH) in the halfcystine solution of various concentration behind the assaying reaction is made absorbancy-concentration standard curve and is calculated the average light absorption value of SH; The working sample solution absorbency, and according to calculating the content that the average light absorption value of SH that obtains is calculated free SH in the sample solution before; The content of free SH is calculated into amount and then the cyclisation degree of polypeptide in the calculation sample solution again of the SH of ring in the solution per sample.
In another aspect of this invention, the invention provides a kind of nucleotide sequence of encode antigenic peptide of the present invention and fused antigen polypeptide.In an embodiment of the invention, the invention provides coding nucleotide sequence of fused antigen polypeptide shown in antigenic peptide and the SEQ ID NOs:35-42 shown in SEQ ID NOs:1-27.
The preparation of polypeptide can realize that it can also can prepare through gene engineering method through the chemical synthesis process preparation through the known technology of this area.Chemical synthesis process mainly comprises solid phase synthesis and the synthetic two kinds of methods of liquid phase.Solid-phase peptide synthesis comprises, Merrifield solid-phase synthesis for example, this method write up document " Merrifield, J.Am.Chem.Soc.85:2149-2154 " and " people such as M.Bodanszky; " In Peptide Synthesis ", John Wiley &Sons, Second Edition; 1976 " and " J.Meienhofer, " Hormonal Proteins and Peptides ", Vol.2; p.46, Academic Press (New York), 1983 ".This by reference mode these documents are incorporated among the application with for referencial use in full.The Merrifield solid-phase synthesis mainly may further comprise the steps, and according to the aminoacid sequence of target polypeptides, protected C-terminal amino acid is connected with resin; Connect the after scouring resin; Slough the protection base (for example, tertbutyloxycarbonyl) on the C-terminal amino acid α amino, slough this protection base time must guarantee not rupture connecting key of this amino acid and interlaminar resin; The protected amino acid of coupling penult C-terminal on the resin of gained then is when carrying out this coupling, at second amino acid whose free carboxy be connected in and form an amido linkage between first amino group of amino acids on the resin; The amino acid order of connection according to target polypeptides repeats the previous reaction process successively, all is connected on the resin up to all amino acid; At last, cut protected peptide, slough the protection base and can obtain target polypeptides from resin.
Polypeptide of the present invention also can for example can be used the solution method of peptide synthesis preparation of standard through liquid-phase synthesis process preparation, this method write up document " E.Schroder and K.Kubke; " The Peptides "; Vol.1, Academic Press (New York), 1965 " in.This by reference mode the document is incorporated among the application with for referencial use in full.Liquid phase synthesizing method mainly comprises, utilizes the chemistry or enzyme method substep coupling amino acid or the peptide fragment that form amido linkage.Gene engineering method is expressed the method that generates corresponding antigens polypeptide or fused antigen polypeptide for the nucleotide sequence that utilizes coding corresponding antigens polypeptide or fused antigen polypeptide in the host cell that is fit to.The detailed description of relevant this method can be with reference to " Sambrook, Fritsch &Maniatis, Molecular Cloning:A laboratory Manual (the 2nd edition, Cold Spring Harbor Laboratory, 1989) ".
According to another aspect of the present invention, the invention provides a kind of vaccine, it comprises foot-and-mouth disease virus antigen polypeptide of the present invention and/or fused antigen polypeptide, and pharmaceutically acceptable vehicle.In one embodiment, vaccine of the present invention comprises multiple foot-and-mouth disease virus antigen polypeptide of the present invention and/or fused antigen polypeptide.In another embodiment, vaccine of the present invention comprises 2 kinds, 4 kinds, 8 kinds, 16 kinds, 32 kinds or 64 kinds of foot-and-mouth disease virus antigen polypeptide of the present invention and/or fused antigen polypeptide.In another embodiment, vaccine of the present invention comprises at least 2 kinds, 4 kinds, 8 kinds, 16 kinds, 32 kinds or 64 kinds of foot-and-mouth disease virus antigen polypeptide of the present invention and/or fused antigen polypeptide.In another embodiment, vaccine of the present invention comprises at least 2 kinds, 4 kinds, 8 kinds, 16 kinds, 32 kinds or the 64 kinds foot-and-mouth disease virus antigen polypeptide that comprise sequence shown in SEQ ID NOs:1-27.
With regard to the present invention, term " pharmaceutically acceptable vehicle " is meant, any suitable pharmaceutically acceptable adjuvant that is used for pharmaceutical prepn, carrier, thinner, sanitas etc.Term " pharmaceutically acceptable " is meant that other composition in corresponding vehicle and the pharmaceutical prepn is complementary at aspects such as chemical property and physicalies, and on physiology, is complementary with the administration individuality, is that the administration individuality can tolerate.The purpose of property as an example only, known adjuvant include but not limited to, for example complete Freund's adjuvant; Incomplete Freund's adjuvant, mineral substance gel are such as white lake, and surfactant is such as the soft phosphatide of haemolysis; Pluronic polyols, polyanion, peptide; Oil-emulsion, the agent of hydrocarbon emulsus, keyhole limpet hemocyanin etc.In an embodiment of the invention, the adjuvant of use is the Montanide ISA series adjuvant that French SEPPIC company provides, and for example Montanide ISA 28; Montanide ISA 035, Montanide ISA 025, Montanide ISA 206; Montanide ISA 575, Montanide ISA 563, Montanide ISA 50V (such as Montanide ISA 50V2); Montanide ISA775, Montanide ISA763A, Montanide ISA70.Known carrier includes but not limited to, sterile liquid, and water for example, oil, the perhaps mixture of water and oil, said oil is peanut oil, VT 18, MO, sesame wet goods for example.Known thinner includes but not limited to, water, salt solution, glucose, ethanol, glycerine and analogue thereof.Known sanitas includes but not limited to, Thiomersalate and EDTA etc.
Pharmaceutically acceptable vectorial selection can realize that the vaccine formulation that those skilled in the art can prepare is as required selected suitable pharmaceutically acceptable vehicle based on prior art through the known technology of this area.For example, for preparation oral liquid (for example, suspension-s, tincture or solution), the vehicle of being selected for use can comprise for example water, oil, alcohols, seasonings, sanitas, tinting material etc.Again for example, for preparation oral solid formulation (for example, pulvis, capsule or tablet), the vehicle of being selected for use can comprise for example starch, carbohydrate, thinner, granulating agent, lubricant, tackiness agent, disintegrating agent etc.In addition, if desired, vaccine of the present invention can also be prepared into sweet tablet or enteric coating, also or controlled release preparation.
Can also be with foot-and-mouth disease virus antigen polypeptide of the present invention and/or fused antigen polypeptide and/or vaccine, be mixed with polyvalent vaccine with other foot-and-mouth disease virus antigen and/or vaccine.For example; OA bivalent vaccine (formulated according to a certain percentage), OC bivalent vaccine (formulated according to a certain percentage), AC bivalent vaccine (formulated according to a certain percentage) and OAC trivalent vaccine (formulated according to a certain percentage) or the like by O serotype foot-and-mouth disease virus antigen, A serotype foot-and-mouth disease virus antigen, C serotype foot-and-mouth disease virus antigen and adjuvant by A serotype foot-and-mouth disease virus antigen, C serotype foot-and-mouth disease virus antigen and adjuvant by O serotype foot-and-mouth disease virus antigen, C serotype foot-and-mouth disease virus antigen and adjuvant by O serotype foot-and-mouth disease virus antigen, A serotype foot-and-mouth disease virus antigen and adjuvant.Other foot-and-mouth disease virus antigen and/or vaccine include but not limited to, other antigenic peptide or fused antigen polypeptide, inactivation of viruses strain, virus antigen, nucleic acid vaccine, immunologic stimulant etc.The polyvalent vaccine that obtains has the immunogenicity of wide spectrum, and it can have immunocompetence to the hoof-and-mouth disease strain and the variant thereof of multiple serotype or multiple hypotype, and the livestock after the polyvalent vaccine immunity can obtain the immunizing power to multiple hoof-and-mouth disease strain.Can also be with foot-and-mouth disease virus antigen polypeptide of the present invention and/or fused antigen polypeptide and/or vaccine; Vaccine with other is mixed with the multi-usage combined vaccine; (see Chen Yingli like foot and mouth disease-swine fever-pseudoabies triple vaccine, foot and mouth disease-brucellosis bigeminy seedling, the sick bigeminy seedling of foot and mouth disease-bubble etc.; Ge Yinsheng, Pan Li.The progress of foot and mouth disease combined immunization.China animal doctor science and technology, 1998,28 (9): 20-21).
According to another aspect of the present invention, the invention provides the method for a kind of control and/or prevention animal foot and mouth disease, this method comprises the antigenic peptide of the present invention that the animal immune of these needs significant quantity is arranged, and/or the fused antigen polypeptide, and/or vaccine.Term " immune significant quantity " is meant the immunoreactive amount that in the administration animal body, causes to foot and mouth disease virus.The immunity significant quantity is relevant with the factors such as species, kind, age, body weight and healthy state of administration animal.In one embodiment, vaccine of the present invention is used for control and/or prevention pig, ox or sheep foot and mouth disease.In another embodiment, vaccine of the present invention is used for control and/or prevention Schweineseuche.In another embodiment, vaccine of the present invention is used to control and/or prevent the O serotype of Schweineseuche.Therefore, the present invention provides a kind of application that antigenic peptide of the present invention and/or fused antigen polypeptide is used for preparing the medicine of control and/or prevention animal foot and mouth disease in yet another aspect.
Vaccine of the present invention can prepare through the conventional medicine compounding process in this area, for example, is mixed into uniform mixture as the antigenic peptide of the present invention of effective constituent and/or fused antigen polypeptide and pharmaceutically acceptable vehicle.Vaccine of the present invention can be through any known route of administration administration, for example subcutaneous, oral, intramuscular or other non-enteron aisles or enteron aisle administration.In an embodiment of the invention, vaccine of the present invention is through subcutaneous or intramuscularly administration.As is known to the person skilled in the art, the consumption of vaccine is according to the difference of factors such as age of the activity of activeconstituents, administration animal individual, body weight and change.Those skilled in the art can easily confirm only vaccine consumption according to the factor of aforementioned affect consumption.
Antigenic peptide of the present invention, fused antigen polypeptide and vaccine can detect its immune efficacy through known several different methods.The TP that prior art provides the multiple assessment vaccine immunity to render a service for example, is attacked malicious protection test, serological test etc.Attack malicious protection test and adopt directly that strong virus is former to carry out the artificial challenge to the experimental animal after the vaccine inoculation, judge the immune efficacy of vaccine then according to the incidence of animal.Attack malicious protection test and be the most directly method that the check vaccine immunity is renderd a service, a plurality of countries that therefore comprise China all require to adopt and attack the immune efficacy that live vaccine is estimated in malicious protection test.Relevant concrete operation method of attacking malicious protection test can be referring to, relevant half protective number (PD in 2010 editions " Chinese veterinary drug allusion quotations " 50) chapters and sections measured.Also can adopt the serological test method to assess the immune efficacy of vaccine, for example measure anti-body contg that produces in the immune serum and then the immune efficacy of judging vaccine through the ELISA method.Normally earlier antigen (or antibody) is combined on the solid phase carrier when adopting ELISA method (being enzyme-linked immunosorbent assay) to measure; Add the conjugate (affinity tag) that a kind of antibody (or antigen) and enzyme are combined into then; After the antigen on conjugate and the solid phase carrier (or antibody) reaction bonded, add the corresponding substrate of enzyme; Promptly play catalytic hydrolysis or redox reaction and be color, the shade that it generated is directly proportional with antigen to be measured (or antibody) content.Have cognation between these serological index and the vaccine potency, it can be used for vaccine immunity effectiveness is carried out entry evaluation.Those skilled in the art can select suitable TP according to factors such as concrete test conditions and test objectives.
According to another aspect of the present invention, the invention provides a kind of method that detects foot and mouth disease virus antibody, this method comprises: one or more antigenic peptides of the present invention or fused antigen polypeptide are provided; This antigenic peptide or fused antigen polypeptide are contacted with specimen, make antigenic peptide or fused antigen polypeptide and foot and mouth disease virus antibodies form antigen-antibody complex; Detect foot and mouth disease virus antibody.Said specimen can be any suitable biological sample from host animal, for example whole blood, serum, blood plasma, also or body fluid.Specimen can be treated (for example dilution), and is also perhaps untreated.
Can realize western blot analysis (Western Blot) for example, perhaps EUSA (ELISA) etc. in the foot and mouth disease virus antibody detection method of the present invention through various known technology for detection of antibodies.USP U.S.Nos.4 discloses the immuno analytical method of many detection antibody in 016,043,4,424,279,4,018,653, these USPs this by reference mode incorporate the application in full into for referencial use.For example; The present invention can use the double-antibody sandwich elisa method to detect foot and mouth disease virus antibody; This method generally includes following steps: capture agent (antigenic peptide for example of the present invention or fused antigen polypeptide) is fixed on the solid substrate, and the specimen (the for example serum of host animal) that will contain target protein (for example foot and mouth disease virus antibody) then contacts with capture agent to form antigen-antibody complex; The detection antibody of detectable reporter molecule that added mark, reaction forms capture agent-target protein-detection antibody complex; Remove unreacted matters, then the signal that produces of examining report molecule and then as qualitative or quantitative analysis.
According to a further aspect of the invention; The invention provides a kind of test kit that detects foot and mouth disease virus antibody; This test kit comprises: a) antigenic peptide of the present invention or fused antigen polypeptide, and, b) can said antigenic peptide or fused antigen polypeptide be fixed the stationary phase on it.Stationary phase is solid substrate normally.Test kit of the present invention can also comprise mark the detectable reporter molecule detection antibody or other can with target protein bonded detection reagent, and/or can with the substrate of examining report molecular reaction.In addition, test kit of the present invention can also further comprise the operation instruction of test kit.
The invention provides foot-and-mouth disease virus antigen polypeptide, fused antigen polypeptide and the vaccine of the synthetic of one type of novelty, it can be used for immune host animal makes it produce the immunizing power that is directed against foot and mouth disease virus widely.Compare with existing foot and mouth disease virus vaccine, antigenic peptide of the present invention, fused antigen polypeptide and vaccine have stronger immunogenicity and immune persistence.It is qualified that the median protective dose of Ministry of Agriculture's regulation foot and mouth disease virus vaccine is greater than 3; The median protective dose of existing foot and mouth disease virus vaccine is all about 6 or lower; And the median protective dose of vaccine of the present invention can be up to more than 11.84; Be up to 15.59, far above the regulation and the existing foot and mouth disease virus vaccine of the Ministry of Agriculture.Antigenic peptide of the present invention, fused antigen polypeptide and vaccine have the immunity of wide spectrum, and for example the different subtype to O type foot and mouth disease virus has extensive applicability.In addition; Because antigenic peptide of the present invention, fused antigen polypeptide and vaccine use peptide sequence not use inactivation of viruses or weak viral disease poison as immunogen; So there is not the spinoff that makes the immune animal infective virus in it, therefore antigenic peptide of the present invention, fused antigen polypeptide and vaccine also have higher security.
Embodiment
Following specific embodiment is further to explain to of the present invention, no matter it all should not be construed as under which kind of situation the present invention is constituted any restriction.
The solid phase synthesis of embodiment 1. polypeptide
Plant and instrument: PSI 300B full-automatic polypeptide synthetic instrument, with Rink Amide mbha resin as solid phase carrier; The positive tangential flow ultrafiltration system of PallCentrasette uses Omega series membranes bag.
Raw material and reagent: the amino acid of 9-fluorenylmethyloxycarbonyl (Fmoc) protection is as starting raw material, other reagent of using in the solid phase synthesis specifically see embodiment with the lower section.All raw materials used in synthetic and reagent can those skilled in the art obtain after simple preparation according to known technology obtaining perhaps through buying pattern on the market.
Through the fused antigen polypeptide (being designated as fused antigen polypeptide 1,2,3,4,5) shown in the synthetic SEQ ID NOs:35-39 of Merrifield solid-phase synthesis.
1, polypeptide is synthetic
The synthetic order of Merrifield solid phase synthesis is to begin to hold to N from the C end.In the good PSI 300B full-automatic polypeptide synthetic instrument of debugging, constantly add the amino acid of Fmoc protection successively and carry out following reactions step successively according to the amino-acid sequence of target polypeptides:
(1) Rink Amide MBHA aminoresin is placed N-Methyl pyrrolidone (NMP) solution that contains 15 ~ 30% (v/v) hexahydropyridine, under 20 ℃ ~ 28 ℃ conditions, reacted 25 ~ 60 minutes, to remove the Fmoc group on the aminoresin;
(2) reacted aminoresin dries up with nitrogen, and with nmp solution washing 6 times;
(3) add I-hydroxybenzotriazole (HOBT), N in the aminoresin after washing, the amino acid of N-DIC (DIC) and first Fmoc radical protection reacted 1 ~ 3 hour under 20 ℃ ~ 28 ℃ conditions then;
(4) add the nmp solution that contains 1.5% ~ 4% (m/v) acetyl imidazole (AIM) to reacted system, reacted 20 ~ 40 minutes down for 20 ℃ ~ 28 ℃ in condition then;
(5) reacted aminoresin dries up with nitrogen, and washs aminoresin 6 times with nmp solution;
(6) repeating step (1)-(5), termination reaction again after all amino acid on the target polypeptides all are connected to aminoresin, it is subsequent use to reclaim reacted aminoresin then.
2, the cracking of polypeptide
It according to the volume ratio of trifluoroacetic acid/phenol/triethyl silicane/water 90/5/4/1 ratio preparation polypeptide lysate.Then aminoresin is mixed with lysate, and ventilate and agitation condition under room temperature successive reaction 1-4 hour.Reaction finishes the back and reclaims reaction product, promptly gets the polypeptide bullion.
3, the purifying of polypeptide
The Rotary Evaporators evaporation polypeptide bullion 30 to 120 minutes that uses the band cold-trap is to remove the trifluoroacetic acid in the bullion.Use the t-butyl methyl ether deposition then and collect polypeptide, and use the t-butyl methyl ether cleaning many times.At last polypeptide is filtered with sand core funnel and drain, promptly obtain pure target polypeptides.
4, the evaluation of polypeptide
The polypeptide that obtains is measured with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF), and its molecular weight is 6132.3 ~ 6676.6.
5, the cyclisation of polypeptide
Use 10%DMSO that polypeptide is configured to the solution of concentration as 2mg/ml, and at room temperature stir with magnetic stirring apparatus.With 1% ammonia soln or 1% acetum polypeptide solution is adjusted to pH value about 6.0 ~ 8.0 then.Every at a distance from 24 hours mensuration pH values 1 time, and according to circumstances regulate pH and make it maintain about 6.0 ~ 8.0 all the time.Continuous cyclization stopped reaction after 192 hours, and collecting reaction product promptly get the cyclisation polypeptide.The cyclisation degree of measuring fused antigen polypeptide 1,2,3,4,5 with the Ellman method of standard is all greater than 87.5%.
6, the repurity of polypeptide
Use strongly basic anion exchange resin that the cyclisation polypeptide is carried out desalting treatment.Use positive tangential flow ultrafiltration system of Pall Centrasette and Omega series membranes to wrap under 20 ℃ ~ 28 ℃ conditions cyclisation polypeptide is carried out ultrafiltration.Performance liquid records the purity of fused antigen polypeptide 1,2,3,4,5 greater than 80%.
7, the degerming of polypeptide
Again in clean work station, using the aperture is that the sterilizing filter of 0.2 μ m carries out filtration sterilization to the cyclisation polypeptide, can obtain aseptic fused antigen polypeptide 1,2,3,4,5 after the purified processing of polypeptide.
The preparation of embodiment 2. antigenic peptide vaccines
1, water preparation
The water for injection that uses sterilising treatment to cross is diluted to 50 μ g/ml with the fused antigen polypeptide 1,2,3,4,5 that embodiment 1 obtains respectively, and the use aperture is the filter filtration of 0.2 μ m.
2, oil phase preparation
Sterilization is subsequent use after 30 minutes down at 120 ℃ for oil phase adjuvant Montanide ISA 50V2.
3, emulsification
Earlier oil phase is added in the emulsion tank, stir with 80 ~ 100r/min then, slowly add water simultaneously; Add oil phase/water ratio be 1/1; Add the back and stirred 2 minutes, stirred 6 minutes with 8500r/min again, make its emulsification form water-in-oil emulsion; Promptly get foot and mouth disease virus vaccine of the present invention, be designated as vaccine 1,2,3,4,5.
Antibody horizontal behind the antigen immune of embodiment 3. various dose detects
The vaccine 1,2,3,4,5 for preparing among test sample: the embodiment 2.
Experimental animal: the healthy feeder pig that foot and mouth disease is negative, the about 40kg/ head of body weight is about 4 monthly ages.
1. TP
Get the vaccine 1,2,3 or 4 of 25 μ g, 8.33 μ g and the preparation of 2.78 μ g fused antigen polypeptide respectively; Each dose groups respectively through 5 pigs of intramuscular inoculation behind the basal part of the ear (20 pigs of vaccine 1 inoculation of 25 μ g dose groups) once; Get the vaccine 5 of 25 μ g fused antigen polypeptide preparation; Respectively through 15 pigs of intramuscular inoculation behind the basal part of the ear; After the 7th, 14,21,28 day, take a blood sample, separation of serum is through the antibody horizontal behind liquid phase blocking-up EUSA (LPB-ELISA) the method detection antigen immune.The ultimate principle of this liquid phase blocking-up EUSA is: with rabbit foot-and-mouth disease virus resistant antibody sandwich elisa plate; The seized serum sample and the fixed amount homology virus antigen mixture of serial dilution are transferred to elisa plate; Add the anti-corresponding serotype foot and mouth disease virus antibody of cavy after the effect; Add the anti-cavy immune globulin antibody of rabbit of enzyme labelling again, the unconjugated reactant of per step flush away is used chromogenic enzyme substrate at last.If in the serum to be checked specific antibody is arranged, then sealed the combination epi-position of enzyme labelled antibody, it is thin out to cause colour developing, the reaction of positive apparent color.
Particularly; This experiment uses foot and mouth disease liquid phase blocking-up ELISA test kit (available from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences) to do above-mentioned liquid phase blocking-up EUSA; According to the experimental procedure operation that the specification sheets of this test kit provides, measure the level of corresponding FMDV antibody in the test serum.Use the negative contrast of O type foot and mouth disease negative serum, the positive contrast of O type foot and mouth disease positive serum.With reading coupling reaction OD value under the 492nm wavelength, judge net result.The concrete operations step is following:
(1) with encapsulating damping fluid dilution foot and mouth disease O type rabbit anti-serum to working concentration (1:1000), every hole adds 50 μ l in elisa plate, shake 2 ~ 3 minutes, then with shrouding film shrouding or put ambient temperature overnight in the box that wets.
(2) with the PBST working fluid serum to be checked is begun with 2 times of seized serum of serial dilution from 1:4 by 50 μ l/ holes amount at U type porous plate; Dilute the negative and positive control serum simultaneously, every then hole adds 50 μ l and is diluted to the O type foot-and-mouth disease virus antigen (1:4) of working concentration with PBST, and the virus antigen control wells adds 100 μ l; The vibration mixing; Shrouding, 4 ℃ are spent the night, and the actual extent of dilution of serum becomes 2 times of serial dilution degree that begin from 1:8 behind the adding virus antigen.
(3) (1 * PBST) washes elisa plate 5 times, on thieving paper, dries, and the antigen antibody reaction plate takes out the room temperature placement after 5 minutes from 4 ℃ with washings; Be transferred to each hole serum-virus mixture in the Sptting plate on the elisa plate in order; Every hole 50 μ l, shrouding was hatched 1 hour for 37 ℃.
(4) the samely wash plate 5 times, dry.To working concentration (1:1000), every hole adds 50 μ l with guinea pig antiserum diluted cavy resisting O-type foot and mouth disease virus serum, and shrouding was hatched 1 hour for 37 ℃.
(5) the samely wash plate 5 times, dry.To working concentration (1:500), every hole adds 50 μ l, hatches 1 hour for 37 ℃ with the anti-cavy immune globulin antibody of PBST dilution rabbit enzyme conjugates.
(6) the samely wash plate 5 times, every hole adds 50 μ l substrate solutions, hatches 15 minutes for 37 ℃.Every hole adds 50 μ l 1.25mol/LH2SO4 termination reactions, under the 492nm wavelength, surveys light absorption value (OD value) immediately.
2. the result judges
Contrasting 50% of average OD value with virus antigen is threshold value; Seized serum dilution holes OD value is greater than negative hole, the hole of threshold value; The positive hole, hole that is less than or equal to threshold value, corresponding extent of dilution was the antibody titers of this part serum when the OD value in positive hole equaled threshold value.If threshold value is between two titres, then antibody titers is got its intermediate value.
ELISA antibody titers and immune animal pig are attacked the relation of poison protection: >=1:64, protection more than 99%;＜1:4 does not protect; 1:4 ~ 1:45,50% protection.
3. test-results
Antibody horizontal detected result behind the vaccine immunity of (1) 25 μ g antigenic peptide preparation:
Figure BDA00002044504200191
Figure BDA00002044504200201
Antibody horizontal detected result behind the vaccine immunity of (2) 8.33 μ g antigenic peptides preparation:
Antibody horizontal detected result behind the vaccine immunity of (3) 2.78 μ g antigenic peptides preparation:
Figure BDA00002044504200231
4. discussion of results
From the result, can find out; Vaccine 1,2,3, the 4 immunity backs of 25 μ g antigenic peptides preparation were risen all to reach more than 99% to protect and (are removed the 14th day animal 2169,2190,2120 and 2155 in the vaccine 1 on the 14th day; Animal 3125 in animal 1561 in the 14th day vaccine 2 and the 14th day vaccine 4 reaches outside 50% protection), the vaccine 5 immunity backs of 25 μ g antigenic peptides preparation were risen all to reach 99% or more and are protected on the 14th day; 8.33 there are three animals (animal 3186 in the vaccine 1, the animal 3161 and 3164 in the vaccine 2) never to reach protection more than 99% after vaccine 1,2,3,4 immunity of μ g antigenic peptide preparation; Other animals all reached a protection more than 99% at least at the 14th, 21 and 28 day; 2.78 after vaccine 1,2,3,4 immunity of μ g antigenic peptide preparation; 8 animals (animal 3168,3169,3170 and 3171 in the vaccine 1 is arranged; Animal 3177,3182 and 3185 in the vaccine 2, the animal 3193 in the vaccine 4) never reached more than 99% and protected; Other animals all reached at least at the 14th, 21 and 28 day one time more than 99% or 50% the protection.This shows that the immune effect of the vaccine of above-mentioned antigenic peptide preparation strengthens with the concentration raising of the antigenic peptide in the vaccine.
The potency test of embodiment 4. polypeptide vaccines
The vaccine 1,2,3,4,5 for preparing among test sample: the embodiment 2.
Experimental animal: the healthy feeder pig that foot and mouth disease is negative, the about 40kg/ head of body weight is about 4 monthly ages.
Test seed culture of viruses: O/0834, O/0718, O/ZK93, O/China99 hoof-and-mouth disease strain (O type), this strain is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.
1, TP
Half protective number (PD according to 2010 editions " Chinese veterinary drug allusion quotation " record 50) measuring method measures respectively the half protective number of vaccine 1,2,3,4,5, concrete grammar is following.Vaccine to be measured is divided into 1 part (1ml vaccine to be measured), 1/3 part (1/3ml vaccine to be measured), 3 dose groups of 1/9 part (1/9ml vaccine to be measured).Each dose groups respectively through 5 pigs of intramuscular inoculation behind the basal part of the ear once.After the vaccine inoculation 28 days, together with 2 of contrast pigs, intramuscular injection 1000 median infective dose (ID behind every pig basal part of the ear 50The O/0834 of)/2ml, O/0718, O/ZK93 or O/China99 hoof-and-mouth disease strain were observed 10 days continuously.Write down the incidence of each treated animal, the animal morbidity promptly is judged to not to be protected.Calculate the percentage of each dose groups immune animal protection.Bubble or ulcer appear in all at least one hoof of contrast pig.The last PD that calculates vaccine 1,2,3,4,5 again according to the Reed-Muench method respectively 50
The Reed-Muench method of calculation are prior arts of this area; Existing document " Reed; L.J.and Muench; H. (1938). " A Simple Method of Estimating Fifty Percent Endpoints " .The American Journal of Hygiene 27:493 – 497 " in this is described in detail, the document this by reference mode incorporate the application into for referencial use.
2, test-results
The PD that table 1. vaccine 1 usefulness O/0834 strain detects 50The result
Figure BDA00002044504200241
Calculate the PD of vaccine 1 according to the Reed-Muench method 50Be 15.59.
The PD that table 2. vaccine 2 usefulness O/0718 strains detect 50The result
Figure BDA00002044504200242
Figure BDA00002044504200251
Calculate the PD of vaccine 2 according to the Reed-Muench method 50Be 11.84.
The PD that table 3. vaccine 3 usefulness O/ZK93 strains detect 50The result
Figure BDA00002044504200252
Calculate the PD of vaccine 3 according to the Reed-Muench method 50Be 13.59.
The PD that table 4. vaccine 4 usefulness O/China99 strains detect 50The result
Figure BDA00002044504200253
Calculate the PD of vaccine 4 according to the Reed-Muench method 50Be 13.97.
The PD that table 5. vaccine 5 usefulness O/0834 strains detect 50The result
Figure BDA00002044504200254
Calculate the PD of vaccine 5 according to the Reed-Muench method 50Be 13.97.
3, discussion of results
Can find out that from table 1,2,3,4,5 vaccine of the present invention can be up to 15.59 for the median protective dose of pig.Median protective dose (the PD of Ministry of Agriculture's regulation aftosa vaccine 50) be greater than 3 qualified, and at present the median protective dose of existing similar foot and mouth disease virus vaccine also all about 6.This shows that the median protective dose of vaccine of the present invention is much larger than the standard of the Ministry of Agriculture, but also far above existing similar vaccine.
Figure IDA00002044504800011
Figure IDA00002044504800021
Figure IDA00002044504800031
Figure IDA00002044504800041
Figure IDA00002044504800051
Figure IDA00002044504800061
Figure IDA00002044504800071
Figure IDA00002044504800101
Figure IDA00002044504800121
Figure IDA00002044504800131
Figure IDA00002044504800151
Figure IDA00002044504800161
Figure IDA00002044504800171
Figure IDA00002044504800181
Figure IDA00002044504800191
Figure IDA00002044504800201
Figure IDA00002044504800211
Figure IDA00002044504800221
Figure IDA00002044504800231
Figure IDA00002044504800241
Figure IDA00002044504800251
Figure IDA00002044504800261
Figure IDA00002044504800281
Figure IDA00002044504800291

Claims (21)

1. foot-and-mouth disease virus antigen polypeptide that comprises aminoacid sequence as follows: Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val Leu X 8X 9Lys Ala Ala Arg X 10Leu Pro Thr Ser Phe Asn TyrX 11Ala Ile Lys (SEQ ID NO:1), wherein, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro, X 10=Thr or Cys, X 11=Gly or Cys, and X 10And X 11Must there be one and can only have one to get Cys.
2. foot-and-mouth disease virus antigen polypeptide as claimed in claim 1 is characterized in that, comprises the aminoacid sequence that is selected from down group: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
3. foot-and-mouth disease virus antigen polypeptide as claimed in claim 1 is characterized in that, comprises aminoacid sequence as follows: Val Tyr Asn Gly X 1Cys Lys Tyr X 2X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val LeuX 8X 9Lys Ala Ala Arg X 10Leu Pro Thr Ser Phe Asn Tyr X 11Ala Ile Lys (SEQ ID NO:8), wherein, X 1=Asn or Asp, X 2=Gly or Ser, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro, X 10=Thr or Cys, X 11=Gly or Cys, and X 10And X 11Must there be one and can only have one to get Cys.
4. foot-and-mouth disease virus antigen polypeptide as claimed in claim 3 is characterized in that, comprises aminoacid sequence as follows: Val Tyr Asn Gly Asn Cys Lys Tyr Gly X 3X 4X 5X 6X 7Asn Val Arg Gly Asp Leu Gln Val LeuX 8X 9Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys (SEQ ID NO:11), wherein, X 3=Glu or Asp, X 4=Ser or Gly, X 5=Pro or Thr or Arg or His, X 6=Val or Thr, X 7=Ala or Thr, X 8=Thr or Ala, X 9=Gln or Pro.
5. foot-and-mouth disease virus antigen polypeptide as claimed in claim 4 is characterized in that, comprises the aminoacid sequence that is selected from down group: SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.
6. like each described foot-and-mouth disease virus antigen polypeptide among the claim 1-5, it is characterized in that said antigenic peptide has the ring texture that forms through disulfide linkage.
7. foot-and-mouth disease virus antigen polypeptide as claimed in claim 6 is characterized in that, the cyclisation degree of said antigenic peptide is at least 85%.
8. a foot and mouth disease virus fused antigen polypeptide is characterized in that, comprises at least two and is selected from each described antigenic peptide among the claim 1-5.
9. foot and mouth disease virus fused antigen polypeptide; It is characterized in that; Comprising at least one is selected among the claim 1-5 each described antigenic peptide and is selected from one or more peptide sequences of following group: t helper cell epi-position, immunostimulatory sequence and natural port fever aphthous virus antigen polypeptide.
10. foot and mouth disease virus fused antigen polypeptide as claimed in claim 9 is characterized in that, said fused antigen polypeptide comprises at least one and is selected from each described antigenic peptide and t helper cell epi-position among the claim 1-5.
11. foot and mouth disease virus fused antigen polypeptide as claimed in claim 10; It is characterized in that said t helper cell epi-position comprises aminoacid sequence as follows: Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val (SEQ ID NO:29).
12. foot and mouth disease virus fused antigen polypeptide as claimed in claim 11 is characterized in that the antigenic peptide of said fused antigen polypeptide is connected by connection chain with the t helper cell epi-position, said connection chain is made up of one or more Lys.
13. foot and mouth disease virus fused antigen polypeptide as claimed in claim 12 is characterized in that, described fused antigen polypeptide comprises the aminoacid sequence that is selected from down group: SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38.
14. foot and mouth disease virus fused antigen polypeptide as claimed in claim 9 is characterized in that said fused antigen polypeptide has the ring texture that forms through disulfide linkage.
15. foot and mouth disease virus fused antigen polypeptide as claimed in claim 14 is characterized in that the cyclisation degree of said fused antigen polypeptide is at least 85%.
16. a nucleotide sequence is characterized in that, each described foot and mouth disease virus fused antigen polypeptide among each the described foot-and-mouth disease virus antigen polypeptide or the claim 8-15 that encodes among the coding claim 1-7.
17. a foot and mouth disease virus vaccine is characterized in that, comprises one or more and is selected from each described antigenic peptide or fused antigen polypeptide and pharmaceutically acceptable vehicle among the claim 1-15.
18. like each described antigenic peptide among the claim 1-7 or like the application of each described fused antigen polypeptide among the claim 8-15 in the medicine of preparation prevention and/or control foot and mouth disease virus.
19. application as claimed in claim 18 is characterized in that, said antigenic peptide is used to prepare the medicine of prevention and/or control pig, ox or sheep foot and mouth disease.
20. a method that detects foot and mouth disease virus antibody, this method comprises: a) provide a kind of like each described antigenic peptide among the claim 1-7 or like each described fused antigen polypeptide among the claim 8-15; B) thus said antigenic peptide or fused antigen polypeptide contacted with specimen form antigen-antibody complex; C) the foot and mouth disease virus antibody in the test sample.
21. a test kit that detects foot and mouth disease virus antibody, this test kit comprises: a) like each described antigenic peptide among the claim 1-7 or like each described fused antigen polypeptide among the claim 8-15; B) can said antigenic peptide or fused antigen polypeptide be fixed the stationary phase on it.
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