CN101838658A

CN101838658A - O type foot-and-mouth disease virus variant as well as coding gene and application thereof

Info

Publication number: CN101838658A
Application number: CN 201010160669
Authority: CN
Inventors: 于力; 杨德成; 于永忠; 周国辉
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2010-04-30
Filing date: 2010-04-30
Publication date: 2010-09-22
Anticipated expiration: 2030-04-30
Also published as: CN101838658B

Abstract

The invention discloses an O type foot-and-mouth disease virus variant as well as a coding gene and application thereof. In the invention, an O type foot-and-mouth disease virus pan-Asia strain O/YS/CHA/05 is firstly separated out, the nucleotide sequence of the O type foot-and-mouth disease virus pan-Asia strain O/YS/CHA/05 SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 2. In comparison with a VP1 amino acid sequence, the virus strain has 7 variable sites, five of which are centralized in a G-H ring. Mutation of the sites ensures that the virus variant has the capability of escaping from host immunity so as to have the superiority for becoming a popular virus strain. Therefore, the variant can be employed to prepare an inactivated vaccine for prevention and treatment of the variant and relevant strains, dominant antigen epitope of the variant can be employed to prepare a synthetic peptide vaccine, and the variant can be employed to develop novel O type foot-and-mouth disease virus vaccines such as VLP vaccine and the like. Therefore, the invention has important value in controlling the popularity of O/YS/CHA/05 and relevant variable strains.

Description

O type foot and mouth disease virus variant and encoding gene and application

Technical field

The present invention relates to O type foot and mouth disease virus, relate in particular to the advantage neutralizing epitope of O type foot and mouth disease virus variant, this O type foot and mouth disease virus variant gene and amino acid sequence coded and this aminoacid sequence, the invention still further relates to this gene, aminoacid sequence or the advantage neutralizing epitope purposes in preparation prevention or treatment O type foot and mouth disease virus vaccine, belong to the prevention and control field of O type foot and mouth disease virus.

Background technology

RNA viruses is because the infidelity and the high speed duplicating of the RNA polymerase that RNA relies on cause RNA viruses to have the heritable variation characteristic of height.The more important thing is that the various progeny virus of this heredity can overcome multiple choices pressure such as host immune and antiviral therapy etc. and be survived, and causes the popular once more of this virus.

Foot and mouth disease is a kind of acute, hot, the height contagious disease that is infected by the artiodactyls such as pig, ox and sheep that foot and mouth disease virus (FMDV) causes.The outburst of foot and mouth disease and popular causes very big loss for local Developing of Animal Industry and foreign trade.Therefore, Food and Argriculture OrganizationFAO and OIE classify it as the No.1 zoonosis that must report.FMDV is the sub-thread positive chain RNA virus, this virus have 7 serotypes (O, A, C, Asia 1, SAT1, SAT2 SAT3), contains a plurality of hypotypes again in each serotype, up to the present existing 65 hypotypes of foot and mouth disease virus.There is not cross protection between each serotype of foot and mouth disease virus; even the antigenic difference degree is also bigger between the different subtype of same serotype; to such an extent as to a kind of vaccine of hypotype may not exclusively be protected the infection of other hypotype FMDV in the same serotype, this has brought great difficulty for the control of foot and mouth disease.At present, the aftosa vaccine that uses of China mainly contains two kinds of inactivated vaccine and synthetic peptide vaccines.

Inactivated vaccine is based on the mixture of the inactivation of viruses of the relevant serotype that contains various reference strains system or hypotype, described reference strain system determines by the virus strain of monitoring localized epidemics, need comprise that the antigen relation between the varient of appearance carries out regular monitoring to wild strain isolated.Need to adopt up-to-date wild strain system to prepare vaccine again, to prevent owing to protection effectiveness is lost in the appearance of new varient.Synthetic peptide vaccine is based on the major antigen site of virus, utilizes the synthetic polypeptide with a fixed structure of a part of aminoacid sequence in viral major antigen site, is used for that immune animal is induced neutralizing antibody and the purpose that reaches protection.If but the sudden change of key amino acid takes place on the main antigen site of virus, will cause the immune protective efficiency of synthetic peptide vaccine to completely lose.The control of China's foot and mouth disease mainly is that vaccine immunity is main.Under strong and lasting immune pressure, the speed of mutation of foot and mouth disease virus is accelerated greatly, constantly brings new challenge for the control of this disease.Monitoring FMDV variation situation has great importance to anti-this disease of system in real time.

In the period of nineteen ninety to 2002 years 12, O type Pan Asia pedigree foot and mouth disease virus is popular in the whole Eurasia, causes heavy economic losses.This strain finds at north India first in nineteen ninety, imports Saudi Arabia in 1994 westwards into, crosses the Middle East in 1996 to import Europe into, is popular in Bhutan in 1998, propagates into most of area in South East Asia the end of the year 1999.Britain broke out nationwide foot and mouth disease and caused and has been very popular March calendar year 2001, imported states such as France, Belgium, Holland subsequently into.Meanwhile, Korea S, Japan and Chinese ground such as Taiwan also successively take place.China 1999 in Tibet, O type Pan Asia pedigree foot and mouth disease takes place in Hainan, a plurality of provinces, Fujian.At Pan Asia foot and mouth disease virus epidemic period, because various countries take to slaughter and extensive immunization strategy makes that this disease is controlled, still monitoring and the control to this virus is the emphasis that various countries prevent and treat foot and mouth disease always.Have research report to show, the Pan Asia strain genome of classics is carried out sequential analysis find that this virus is in the period of popular 12, primary structure albumen VP1 does not almost morph.But, in recent years, be that the master prevents and treats the country of foot and mouth disease with the immunization strategy at some, Pan Asia correlated virus and Pan Asia 2 type strains have appearred in the report that constantly has the Pan Asia foot and mouth disease virus to morph.

Summary of the invention

One of purpose of the present invention provides the variant of a strain O type Pan Asia pedigree foot and mouth disease virus;

Two of purpose of the present invention provides the genome and the amino acid sequence coded thereof of above-mentioned O type Pan Asia pedigree foot and mouth disease virus variant;

Three of purpose of the present invention provides the major antigen site of above-mentioned O type Pan Asia pedigree foot and mouth disease virus variant;

Four of purpose of the present invention is that described O type Pan Asia pedigree foot and mouth disease virus variant and major antigen site are applied to prepare the vaccine that can prevent or treat O type Pan Asia pedigree foot and mouth disease virus variant;

The present invention seeks to be achieved through the following technical solutions:

The present invention at first is separated to a strain Pan Asia pedigree foot and mouth disease virus O/YS/CHA/05; Its separation method comprises: get the morbidity swine disease and become tissue sample (blister fluid and/or bubble skin), place the sterile tissue mill to grind, add a small amount of PBS and microbiotic, make suspension.Continuing to add PBS in the grinding is 10 times of epithelium sample until cumulative volume, is made into 10% suspension.Behind twice of the multigelation,, get supernatant with desk centrifuge 2000g centrifugal 10min, behind the membrane filtration with 0.22 μ m, inoculation individual layer BHK-21 cell, treat to receive after the cytopathy malicious, behind the multigelation three times, preserve-70 ℃ standby.

Because foot and mouth disease virus belongs to the animal pathogenic microorganism of high risk, for this reason, the present invention with the infective cloned plasmids (pOKT7-O/YS/CHA/05) of isolating Pan Asia pedigree foot and mouth disease virus O/YS/CHA/05 strain submit the preservation of patent accreditation body to, its microbial preservation number is: CGMCC No.3719; The classification name is: colon bacillus (Escherichia coli); The preservation time: on April 9th, 2010; Depositary institution is: Chinese common micro-organisms culture presevation administrative center; The preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; This plasmid can be obtained O/YS/CHA/05 virus through in-vitro transcription and transfection BHK-21 cell, concrete in-vitro transcription method is please carried out according to the embodiment of the invention 1.5 described in-vitro transcription methods, and described transfection can be carried out according to the embodiment of the invention 1.6 described transfection methods.

The comparison of VP1 aminoacid sequence finds that this sudden change strain has 7 variant sites, and wherein 5 variant sites concentrate in the G-H ring.The present invention has carried out complete sequence determination with this Pan Asia pedigree foot and mouth disease virus O/YS/CHA/05 strain, and its nucleotides sequence is classified as shown in the SEQ ID NO:1, and its amino acid sequence coded is shown in the SEQ ID NO:2.

Antigenic variation and molecular evolution rule for research Pan Asia virus, the present invention has made up reverse genetic manipulation platform that should virus and has carried out the reverse reverting sudden change at 5 variant sites of the above-mentioned G-H of being positioned at intra-annular, with the antigenic molecular evolution rule of retrospective study Pan Asia pedigree FMDV.Utilize one group of monoclonal antibody to analyze the antigenicity of the mutated viruses of these rescues, find that wherein strain recombination mutation virus V-P153Q is no longer discerned by strong neutrality monoclonal antibody 1F6 at O/YS/CHA/05 virus.The epi-position of utilizing the fusogenic peptide scanning technique to determine that monoclonal antibody 1F6 is discerned is ¹⁴⁷DLQVLTPK ¹⁵⁴, it is positioned at the proteic G-H ring of O type FMDV VP1, the advantage neutralizing epitope 8E8 epi-position core that an O type FMDV of this epi-position and inventor's laboratory qualification in the past is conservative ( ¹⁴⁷DLQVLT ¹⁵²) overlapped.Utilize the L-Ala scanning method, the present invention has identified the key amino acid of MAb 8E8 and MAb 1F6 identification epi-position.8E8 epi-position key amino acid is D ¹⁴⁷, Q ¹⁴⁹, V ¹⁵⁰Residue, 1F6 epi-position key amino acid is D ¹⁴⁷, P ¹⁵³Residue.The proteic aminoacid sequence compare of analysis of the corresponding VP1 of 1F6 epi-position shows T in this epi-position ¹⁵², P ¹⁵³Be the variant sites of this strain, other O type strain mainly is A in this position ¹⁵², Q ¹⁵³For further analyzing these two influences that site mutation sexually revises this virus antigen, the present invention has made up the recombinant virus v-TP/AQ of a strain combinatorial mutagenesis again, to detect independent sudden change in two sites and combinatorial mutagenesis to neutrality monoclonal antibody 1F6, the influence of the neutralising capacity of 8E8 and mouse-anti totivirus serum, the result shows, monoclonal antibody 1F6 is to v-P153Q, v-TP/AQ neutralization is tired and is dropped to less than 4 by original 4096, monoclonal antibody 8E8 drops to 256 to the v-TP/AQ neutralising capacity by original 8192, and mouse totivirus serum drops to 22 to the neutralising capacity of v-TP/AQ by original 355.The result shows; these two site mutation have changed viral antigenic structure to a great extent; cause an advantage neutralizing epitope to disappear; another advantage neutralizing epitope significantly weakens; active being close to of the sero-fast neutralization of totivirus disappears; this will cause existing O type foot and mouth disease inactivated vaccine to the approaching forfeiture of the protection of variant, and the protection of synthetic peptide vaccine completely loses.Therefore, upgrade the vaccine kind poison and the existing synthetic peptide vaccine of regenerating again in time to preventing that being very popular of this variant from being necessary.

Up to the present, O type FMDV has been shown to contain five antigen sites, and wherein the G-H ring is that site A is the major antigen site of FMDV.There are some researches show 50% antibody is arranged in the antibody that vaccine immunity produces at this site.In addition, site A exists a plurality of successive epi-positions, and a site mutation will cause the active monoclonal antibody of a plurality of neutralizations to lose the neutralization activity.Therefore, the sudden change of indivedual key amino acids all can cause the generation of variant in this site.Result of study of the present invention shows, 2 of VP1 protein 15s and any one site mutation of 153 amino acids all will influence the neutralization activity of relevant neutralizing antibody, and these two site simultaneous mutations have significant Overlay.

The present invention shows by above-mentioned great deal of experimental, the present invention isolating O/YS/CHA/05 virus be the new variant of O type Pan Asia pedigree foot and mouth disease virus, the immunne response of existing vaccine can be escaped basically, being very popular of foot and mouth disease virus of a new round might be caused.Utilize strain of the present invention and relevant strain thereof to prepare vaccine, or preparation contains the 8E8 that the inventor identifies and the synthetic peptide vaccine or the combined vaccine of two neutralizing epitopes of 1F6, can more effectively prevent and make the popular of control foot and mouth disease.

Description of drawings

The genetic evolution tree of Fig. 1 VP1 gene.

Fig. 2 O type FMDV O/YS/CHA/05 pnca gene group is spliced tactful synoptic diagram.

The structure (B) of Fig. 3 O type FMDV O/YS/CHA/05 strain antigen site aminoacid sequence comparison (A) and recombination mutation virus.

The suddenly change antigenicity analysis of recombinant virus of Fig. 4.

Among Fig. 5 monoclonal antibody 8E8 and the 1F6 and specific activity.

Fig. 6 monoclonal antibody 8E8 identification epi-position maximum activity unit is identified.

Fig. 7 monoclonal antibody 8E8 and 1F6 variable region are light, the heavy chain amino acid sequence comparison.

The accurate location of Fig. 8 monoclonal antibody 1F6 identification epi-position.

The key amino acid of Fig. 9 monoclonal antibody 8E8 and 1F6 identification epi-position.

The external checking of Figure 10 T ¹⁵², P ¹⁵³Sudden change to monoclonal antibody 8E8 and 1F6 in conjunction with active influence.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.

Embodiment

1, experiment material and method

1.1 cell, virus and antibody

BHK-21 cell (purchasing the ATCC in the U.S.), nutrient solution is the DMEM that contains 10% foetal calf serum, is containing 5%CO ₂37 ℃ of incubators in cultivate.Isolating O type foot and mouth disease virus O/YS/CHA/05 strain in containing the DMEM nutrient solution of 2% foetal calf serum, breed, results virus when CPE appears in the cell when 80%, behind the multigelation three times, 2000 * g, (4 ℃) centrifugal 10min, supernatant are stored in-70 ℃.Intestinal bacteria competence DH5a and BL21 (DE3) are preserved by this laboratory, and the pGEX-6p-1 carrier is purchased the company in Promega.The pOK12 carrier is so kind as to give (the GenBank accession number is AF223639) by Messing, and the present invention has removed the T7 promoter sequence of this carrier with EcoRV and FspI double digestion.

(microbial preservation of the hybridoma cell line of secrete monoclonal antibody 8E8 number is: CGMCC 2691 for the monoclonal antibody 8E8 of O type FMDV O/YS/CHA/05 virus strain; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: Datun Road, Chaoyang District, Beijing City; ), (microbial preservation of the hybridoma cell line of secrete monoclonal antibody 4B2 number is: CGMCC No.3103 for 1F6 (available from biotechnology development company of Harbin dimension section), 5F7,4A8,2B9,4B2; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: Datun Road, Chaoyang District, Beijing City.5F7,4A8 and 2B9 are available from biotechnology development company of Harbin dimension section).

1.2 agents useful for same and Restriction Enzyme

PrimeSTAR archaeal dna polymerase, Primerscript II ThermoScript II, various restriction enzyme are all available from TaKaRa company.T ₄Dna ligase is available from NEB company.TRIzol is available from Gibco BRL company.In-vitro transcription adopts the RiboMAX of Promega company ^TMLarge Scale RNA ProductionSystems-T7 test kit.Transfection reagent adopts QIAGEN company

Transfection Reagent transfection reagent box.

The sheep anti-mouse igg fluorescence antibody is available from Sigma company.

1.3 the extraction of viral RNA, RT-PCR

Extract viral RNA according to TRIzol reagent (Gibco BRL) product description, use Oligod (T)-15 to carry out reverse transcription then, with this reverse transcription product is template, full genome with table 1 primer segmentation amplification O/YS/CHA/05 virus, and glue recovery PCR product, send the order-checking of Invitrogen company.And (TaKaRa, Dalian) operation instruction are RACE to 5 of virus ' end, check order, thereby record virus genome complete sequence accurately to press 5 ' RACE test kit.In addition, for making up the O/YS/CHA/05 virus infection clones, with four overlapping fragmentses of A, B, C, D of primer amplification covering gene group total length in the table 2.Introduce the t7 rna polymerase promoter sequence at viral genome 5 ' end simultaneously, introduce EcoRV restriction enzyme restriction enzyme site at 3 of virus ' end.Use site-directed mutagenesis technique, C is sported T, make this position AGATCT (XbaI) become AGATTT, introduce molecular label by eliminating the XbaI enzyme cutting site in viral genome 3510nt position.

The sequencing primer of table 1FMDV O/YS/CHA/05 strain full-length gene group

Table 2 makes up the primer of FMDV O/YS/CHA/05 strain infections clone and recombinant virus

1.4 the structure of recombinant virus

G-H encircles interior 5 influences that the mutational site sexually revises this virus antigen for research O/YS/CHA/05 virus, and the present invention uses rite-directed mutagenesis test kit (Invitrogen) common property on the O/YS/CHA/05 infections clone basis that has built to give birth to 6 virus mutant v-K138E, v-A142T, v-A144V, v-T152A, v-P153Q, v-TP/AQ.On the G-H ring, do specific amino acid sudden change the primer and see Table 2.Use the used plasmid of plasmid extraction kit (QIAGEN) preparation in-vitro transcription.The recombinant vectors of all structures checks order through total length, does not have other any sudden change.

1.5 in-vitro transcription

Adopt RiboMAXTM Large Scale RNA Production SystemsT7 system to carry out in-vitro transcription, reaction system is: 25mmol/L rNTP 6 μ L, 5 * damping fluid, 4 μ L, t7 rna polymerase mixed solution 2 μ L, linearizing pOKT7-O/YS/CHA/05 recombinant plasmid of EcoR V and mutated viruses recombinant plasmid 8ul (2 μ g), cumulative volume is 20 μ L.Behind the abundant mixing of reactant,,, press the RNA of phenol chloroform method for extracting purifying in-vitro transcription with RNase-Free DNase dna digestion template in 37 ℃ of incubation 2-4h.

1.6 transfection

When the BHK-21 cell grows to the 70%-90% individual layer in 6 orifice plates, wash cell twice, add 1.5mL normal cell nutrient solution with PBS.RNA difference transfection BHK-21 cell with the in-vitro transcription acquisition.QIAGEN company is pressed in transfection

Transfection Reagent transfection reagent box specification sheets carries out, and cells transfected is at 5%CO ₂Incubator in 37 ℃ of incubation 4h, change substratum with the DMEM that contains 2% foetal calf serum and continue to cultivate, the observation of cell pathology, about about 3 days results virus is inoculated the BHK-21 cell behind the multigelation 3 times once more, can stablize generation CPE up to virus.

1.7 indirect immunofluorescence

After being grown in the BHK-21 cell infection virus 6h in 96 orifice plates, with the PBS rinsing 2 times and the raffinate that exhausts, with the fixing 15min of-20 ℃ of cold dehydrated alcohols, drip monoclonal antibody (8E8,1F6,5F7,4A8,2B9, the 4B2) ascites of dilution in 1: 100 respectively, incubation 60min in 37 ℃ of wet boxes with PBS rinsing 5 times, adds FITC sheep anti-mouse igg (1: 100) incubation 45min in 37 ℃ of wet boxes, with 70% glycerine mounting, under the 0lympus fluorescent microscope, observe and take pictures.

1.8GST Expression of Fusion Protein

For determining the minimum unit of monoclonal antibody 1F6 identification epi-position, a series of expression VP1 peptide sections ¹⁴⁶GDLQVLTPKAART ¹⁵⁸And the complementary oligonucleotide chain of truncated peptide is synthesized (seeing Table 3), and the annealing of complementary oligonucleotide chain warp can form the sticky end of restriction enzyme BamHI and XhoI, thereby is inserted among the prokaryotic expression carrier pGEX-6p-1.The carrier transformed into escherichia coli BL21 (DE3) that builds, the picking positive colony, the IPTG that adds final concentration then and be 1mmol/L carries out abduction delivering.In addition, for identifying key amino acid in monoclonal antibody 1F6 and the 8E8 epi-position, utilize the L-Ala method for scanning, replace the amino acid in the 1F6 epi-position one by one, every mutant peptide is expressed in the gst fusion protein mode equally.The complementary oligonucleotide chain of expressing mutant peptide sees Table 3.

The oligonucleotide of the small peptide that table 3 coding and GST albumen merge

1.9SDS-PAGE and Western blot analyzes

Approximately the gst fusion protein sample of equivalent carries out SDS-PAGE electrophoresis and Western blot analysis.Transfer printing and sealing behind the electrophoresis, the odd contradictive hydroperitoneum (1: 1000) with the confining liquid dilution reacts 1h for 37 ℃ again; With Tris-HCl washing lotion washing 3 times, each 10min is then with the sheep anti-mouse igg room temperature reaction 1h of 1: 5000 times of dilution, HRP mark; Wash 3 times, each 10min is at last with the colour developing of DAB substrate colour developing liquid, observations.

1.10 viral microneutralization test

Measure the TCID50 of the mutated viruses of FMDV O/YS/CHA/05 and each rescue according to a conventional method with the BHK-21 cell, adopt fixed virus dilution antibody (monoclonal antibody 8E8,1F6,4B2 and mouse-anti totivirus serum) method to carry out micro-cell neutralization test: to select different with the equal-volume respectively dilution antibody of virus of 100TCID50 to mix 37 ℃ of water-bath 1h; Then above-mentioned antibody-viral mixed solution is inoculated the BHK-21 cell monolayer respectively, every hole 100 μ L, every titre is established 8 holes and is repeated, in 37 ℃ of 5%CO ₂Cultivate observation of cell every day in the incubator.In addition, establish the contrast of viral ascites and normal cell, press the Reed-Muench method according to cytopathic effect (CPE) and calculate in the virus and titre, can protect the 50%BHK-21 cell ascites weaker concn of CPE not occur.

1.11 addition elisa assay

Measure additivity index with addition ELISA.With the suitableeest antigen concentration coated elisa plate, add each strain monoclonal antibody purification of saturation concentration and equal-volume blended monoclonal antibody 100 μ L in twos behind the thorough washing respectively, every strain repeats 2 holes, incubates 1h for 37 ℃; The washing back adds the HRP-sheep anti-mouse igg, hatches 1h for 37 ℃; OD is measured in the colour developing of washing back respectively ₄₉₂Value.Be calculated as follows the AI value after each strain monoclonal antibody superposes in twos: AI=(A1.2-A1)/A2 * 100%.A1.2 is the OD after 2 strain monoclonal antibodies superpose in twos in the formula ₄₉₂Value; A1 is the OD of the 1st strain monoclonal antibody ₄₉₂Value; A2 is the OD of the 2nd strain monoclonal antibody ₄₉₂Value.When the synergetic AI value of antibody in twos greater than 10% the time, show this 2 strain antibody institute identified epitope difference; When the synergetic AI value of antibody in twos less than 10% the time, show that this 2 strain antibody institute identified epitope is identical or close.

1.12 indirect ELISA

Be diluted to 20 μ g/mL after the gst fusion protein of L-Ala scanning mutant peptide is purified, add enzyme plate (every hole 100 μ L), 4 ℃ are spent the night; Wash 3 times with PBS, the sealing of 5% skimming milk is hatched 1h for 37 ℃; Wash 3 times with PB, add monoclonal antibody 8E8 or 1F6, hatch 1h for 37 ℃; The HRP mark sheep anti mouse two that the washing back adds dilution in 1: 5000 resists, and the OPD colour developing is read the OD value in 492nm.

2, experimental result

2.1, O type FMDV O/YS/CHA/05 strain whole genome sequence measures

For recording the viral accurately full-length gene group of FMDV O/YS/CHA/05 strain, the present invention adopts the PCR product directly to check order and 5 of virus ' end is carried out 5 ' RACE analyze, thereby guarantees that the complete genomic 5 ' end of O/YS/CHA/05 strain that we record also is real.O/YS/CHA/05 genome total length 8182nt (SEQ ID NO:1), this strain of VP1 evolutionary tree analysis revealed belongs to O type FMDV Pan Asia pedigree (as Fig. 1).

2.2, the foundation of FMDV O/YS/CHA/05 strain reverse genetic service platform

Genome sequencing result according to FMDV O/YS/CHA/05 strain analyzes available restriction enzyme site, design splicing strategy (Fig. 2).With 4 overlapping fragmentses of A, B, C, D of covering gene group total length, by 3 ' hold 5 ' end in turn splicing clone in pOK12 carrier through transforming, obtain pOKT7-O/YS/CHA/05.Meet desired design fully through the constructed full length cDNA sequence of sequencing checking, promptly 5 ' end is introduced the T7 promoter sequence, Poly (C) comprises poly (A) sequence that 16 C bases, 3 ' end contains 16nt.Simultaneously, the XbaI restriction enzyme site of 3510 positions is eliminated to the replacement of T by base C in the FMDV infections clone genome, thereby can be used as molecule marker with the difference parental virus.

2.3, the rescue of recombinant virus

Isolating strain O/YS/CHA/05 of the present invention and the comparison of Pan Asia classical strains VP1 aminoacid sequence are found (Fig. 3 A), have 7 amino-acid residues and undergo mutation that wherein 5 sudden changes are positioned at the G-H ring.The G-H ring is the major antigen site of foot and mouth disease virus, is the antigen position of inducing protection antibody, also is the combining site of foot and mouth disease virus cell receptor simultaneously.The amino acid at this position easily makes a variation, and is the main mode and the approach of viral escape host immune response.For studying the effect of the amino acid whose variation in this site in the foot-and-mouth disease virus antigen evolutionary process, and it is whether relevant with the viral escape host immune, the present invention utilizes the reverse genetic technology that the reverse mutation of retrospective has been carried out in these variations, has saved 5 plant mutant viruses (Fig. 3 B).

2.4, the antigenicity analysis of recombinant virus

Use one group of monoclonal antibody (8E8 at the O/YS/CHA/05 strain, 1F6,5F7,4A8,2B9,4B2), utilize indirect immunofluorescence method that this 5 strain recombinant virus antigens is analyzed (Fig. 4), find wherein strain recombination mutation virus V-P153Q and monoclonal antibody 8E8,5F7,4A8,2B9, the 4B2 reaction is normal, but lost and neutrality monoclonal antibody 1F6 bonded ability, the sudden change that shows this amino-acid residue destroyed 1F6 at epi-position, may be relevant with the antigenic variation of virus, therefore monoclonal antibody 1F6 and this site mutation have been done further analysis to the influence of virus antigenicity.

2.5, the characteristic of neutrality monoclonal antibody 1F6 and 8E8

As antigen immune Balb/c mouse, the preparation monoclonal antibody has obtained hybridoma 8E8 and 1F6 that two strains can be secreted persistent erection and active monoclonal antibody with the O/YS/CHA/05 totivirus of deactivation.Carry out subgroup identification after antibody is purified, 1F6 heavy chain type is IgG2b, and 8E8 heavy chain type is IgG2a, and the two light chain type is the kappa chain.It is 1: 4096 that 1F6 odd contradictive hydroperitoneum neutralization is tired, and it is 1: 8192 that the neutralization of monoclonal antibody 8E8 ascites is tired, behind the purifying two strain monoclonal antibodies in and potency ratio the results are shown in Figure 5.Stack ELISA experimental result shows the two stack AI less than 5%, shows that monoclonal antibody 1F6 is identical or close with 8E8 identification epi-position.In addition, the identification of this two strains monoclonal antibody all are linear epitopes because Western blot result show the two all can with the O/YS/CHA/05 viral capsid proteins reaction of sex change.Inventor laboratory had identified that monoclonal antibody 8E8 identification epi-position minimum unit was positioned on the O type FMDV VP1 albumen in the past, and the identification motif is ¹⁴⁷DLQVLT ¹⁵², the present invention identifies that further the maximum activity unit of monoclonal antibody 8E8 identification epi-position is ¹⁴⁵RGDLQVLTPK ¹⁵⁴(see figure 6).

To 8E8 and 1F6 two strain antibody mabs are light, variable region of heavy chain checks order and the aminoacid sequence of deriving, the two aminoacid sequence comparison result is seen Fig. 7.The two compares evident difference is that monoclonal antibody 8E8 lacks 5 amino acid in the CDR1 district of variable region of light chain.And at antibody heavy chain variable region, monoclonal antibody 1F6 is at 3 amino acid of CDR3 district disappearance.These results show that the epi-position of monoclonal antibody 1F6 and 8E8 identification is close and inequality.

2.6, the accurate location of 1F6 identification epi-position

Because monoclonal antibody 1F6 can not react with mutated viruses v-P153Q, the epi-position of therefore inferring monoclonal antibody 1F6 identification is near the proteic 153 amino acids residues of VP1.Utilize pepscan, the 140-158 peptide of a series of brachymemmas is carried out the reaction (Fig. 8) of GST amalgamation and expression and usefulness Western blot method detection monoclonal antibody 1F6 and gst fusion protein.The result shows, ¹⁴⁷DLQVLTPK ¹⁵⁴Be the minimum unit of monoclonal antibody 1F6 identification epi-position, this epi-position and the 8E8 epi-position core of identifying in the past ¹⁴⁷DLQVLT ¹⁵²Overlapping.

2.7, L-Ala scanning determines 1F6 epi-position and 8E8 epi-position key amino acid

For understanding the influence to virus antigenicity of interaction between the Ag-Ab and the indivedual amino acid mutations of epitope regions better, the present invention utilizes the L-Ala method for scanning, replaces 1F6 epi-position amino acid one by one with L-Ala.And carry out the GST amalgamation and expression.After gst fusion protein was purified, bag was detected each mutant peptide with indirect ELISA method and combines active influence (Fig. 9) with MAb 8E8 and MAb 1F6 by 96 hole enzyme plates.The result shows, compares in conjunction with activity with original 1F6 epi-position with MAb 1F6, and mutant peptide D147A, P153A combine activity with MAb 1F6 and all descend more than 75%, shows that 147 and 153 amino acids residues are key amino acids of this epi-position.Mutant peptide Q149A, V150A, K154A combine with MAb 1F6 activity descend also more obvious, show these three amino-acid residues with the combining of MAb 1F6 in also play an important role.In addition, compare with original peptide, mutant peptide D147A, Q149A, V150A combine activity with MAb 8E8 and all descend more than 80%, show that 147,149,150 amino acids residues are key amino acids of 8E8 epi-position.Residue D wherein ¹⁴⁷It is the key amino acid that these two overlapping epi-positions are shared.

2.8, two site mutations of vitro detection epi-position to monoclonal antibody 8E8 and 1F6 in conjunction with active influence

Find that by O type FMDV VP1 aminoacid sequence is compared 152 Threonines and 153 proline(Pro) are variant sites in the 1F6 epi-position, and other strains are L-Ala (minority is a Threonine) at 152, are glutamine at 153.For disclose these two site mutation to two strain persistent erections and active monoclonal antibody in conjunction with active influence, we are at external sudden change and these two amino acid of simultaneous mutation respectively, and express the gst fusion protein of mutant peptide.The detected (see figure 10) of fusion rotein of about equivalent, Western blot analysis revealed, mutant peptide T152A and P153Q all obviously descend with the activity that combines of MAb 1F6, and the P153Q sudden change is more obvious in conjunction with activity for reducing MAb 1F6 than the T152A sudden change, causes the binding ability forfeiture of this mutant peptide and MAb 1F6 when two amino-acid residue simultaneous mutations.T152A sudden change equally also influences the combination of MAb 8E8, and the P153Q sudden change does not change the activity that combine with MAb 8E8.

2.9, detect two site mutations in the body to the antigenic influence of virus O/YS/CHA/05

For further studying T ¹⁵², P ¹⁵³These two sudden changes are to the influence of O/YS/CHA/05 virus antigenicity, the present invention has made up and has saved the recombinant virus (V-TP/AQ) of these two site simultaneous mutations again, in conjunction with the unit point mutated viruses of saving previously (V-T152A and V-P153Q), utilize viral microneutralization test method, the antigenic influence degree of this dibit point mutation virus neutralization is detected (Figure 11).At first, it is 1: 4096 that monoclonal antibody 1F6 ascites is tired to the neutralization of virus, descends 2.4 times and the neutralization of T152A mutant strain tired, and the neutralization activity of mutant strain P153Q and TP/AQ is completely lost.For monoclonal antibody 8E8, the T152A sudden change makes among the MAb8E8 and active decline 2.6 times, and the P153Q sudden change makes among the MAb 8E8 and active decline 4 times, and two site simultaneous mutations (TP-AQ) have tangible Overlay, make among the MAb 8E8 and active decline 32 times.For anti-totivirus serum, these two site mutation have same effect, totivirus serum neutralization is tired descend 16 times, drop to 22 by original 355.Above experimental data shows that O/YS/CHA/05 virus is escaped the antigenic variation strain for neutralization.

Detect T in table 4 body ¹⁵², P ¹⁵³Sudden change is to the influence of O/YS/CHA/05 virus antigenicity

Sequence table

＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture

＜120〉O type foot and mouth disease virus variant and encoding gene thereof and application

<130>KLPI0768

<160>2

<170>PatentIn?version?3.1

<210>1

<211>8262

<212>DNA

<213>Hand-foot-and-mouth?disease?virus

<400>1

ttgaaagggg?gcgttagggt?ctcatcccta?acacgccaac?gacagctcct?gcgctgcact 60

ctacactcac?gtctgtgtgc?gcgcggggac?cgttggacta?ccgttcaccc?acctacggtt 120

ggactcacgg?caccgcgcgg?ccattttagc?tggattgtgt?ggacgaacgc?cacttgcgca 180

ctccgcgtga?ctggttaata?ctcttaccac?tttccgccta?cctggtcgtt?ggcgctgtcc 240

tgggcactcc?cgttgggggc?cgtccggtgc?tccacggttt?ccacgcgtga?caaactacgg 300

tgatggagcc?gcttcgtgcg?agttgatcgc?ctggtgtgtt?tcggctgtca?cccgaagtcc 360

acctttcacc?cccccccccc?ccccccccca?cctctcacaa?gtttttaccg?cctttcccag 420

cgttaaaggg?aggtaaccac?aagcttgcgt?ctgtcttgct?cgacgataaa?gggctgtgac 480

cgcaagatga?taccgccttt?cccggcgtta?attggatgta?accataagac?gaaccttcac 540

ccggaagtaa?aacggcaaat?tcgcatagtt?ttgcccgttt?tcaggagaaa?cgggacgtct 600

gcgcacgaaa?cgcgccgtcg?cttgaggagg?acttgtacaa?acacggtcca?ttcaggtttc 660

cacaaccgac?acaaaccgtg?caacttggaa?ctccgcctgg?tctttccagg?tctagagggg 720

tgacattttg?tactgtgctt?gactccacgc?tcggtccact?ggcgagtgct?agtaacagca 780

ctgttgcttc?gtagcggagc?atggtggccg?cgggaactcc?tccttggtaa?cagggacccg 840

cggggccgaa?agccacgtcc?tcacggaccc?accatgtgtg?caaccccagc?acggcaactt 900

tattgtgaaa?accactttaa?ggtgacactg?atactggtac?tcaaccactg?gtgacaggct 960

aaggatgccc?ttcaggtacc?ccgaggtaac?acgcgacact?cgggatctga?gaaggggact 1020

ggggcttctt?taaaagcgcc?tggtttaaaa?agcttctacg?cctgaatagg?tgaccggagg 1080

ccggcacctt?tccttcgaac?aactgtcttt?aaatgagcac?aactgactgt?ttcatcgctt 1140

tgttgtacgc?tttcagagag?atcaaaacac?tgtttttatc?acgaacgcga?ggaaagatgg 1200

agttcacact?tcacaacggt?gagaagaaaa?cattctactc?cagacccaac?aaccacgaca 1260

actgctggtt?gaacgccatc?ctccagctgt?ttaggtacgt?tgatgaacct?ttcttcgact 1320

gggtctactg?ttcacacgag?aacctcacac?tcaatgctat?aaaacaattg?gaagaaatta 1380

ctggtctcga?gctccacgag?ggtggaccac?ccgctctcgt?tatttggaac?atcaaacacc 1440

tgctcaacac?cggaataggc?accgcttcgc?gacccagcga?agtgtgcatg?gtagacggga 1500

cggacatgtg?cttggctgac?ttccatgctg?gcatcttcct?gcaaggacag?gaacacgctg 1560

tgttcgcctg?cgtcacctcc?aacgggtggt?acgcaatcga?tgacgaggac?ttttacccct 1620

ggacgccgga?cccgtccgac?gttctggtgt?tcgtcccgta?cgaccaagaa?ccgctcaacg 1680

gagaatggaa?aacaaaggtt?cagaaacgac?tcagaggtgc?cgggcaatcc?agcccggcga 1740

ctgggtcgca?gaaccagtca?ggtaacactg?gaagcattat?caacaattac?tacatgcagc 1800

agtaccagaa?ctccatggac?acacaacttg?gtgacaacgc?tattagtgga?ggctccaacg 1860

aggggtccac?ggacaccacc?tccacccaca?caaccaacac?ccaaaacaac?gactggtttt 1920

caaagctagc?cagttctgct?tttagcggtc?ttttcggcgc?tcttctcgct?gacaagaaaa 1980

ccgaggagac?cactcttctt?gaggaccgca?tcctcactac?ccgcaacggg?cacacgacct 2040

cgacaaccca?gtcaagcgtt?ggagtcactt?acgggtacgc?gacagctgag?gactttgtga 2100

gcggaccgaa?cacgtctggg?cttgagacca?gggttgtgca?ggcagagcgg?ttcttcaaaa 2160

cccacttgtt?cgactgggtc?accagtgacc?cgttcggacg?gtgctacctg?ctggaactcc 2220

caactgacca?caaaggtgtc?tacggtagcc?taactgactc?ttatgcttac?atgagaaacg 2280

gttgggatgt?agaggttact?gcagtgggga?atcagttcaa?cggaggatgt?ctgttggtgg 2340

ctatggtacc?agaactttgc?tctattgaca?agagagggct?ttaccaactc?acgctcttcc 2400

cccaccagtt?catcaacccc?cggacgaaca?tgacggcgca?catcactgtg?ccttttgttg 2460

gcgtcaaccg?ctacgaccag?tacaaggtac?acagaccttg?gactctcgtg?gtcatggttg 2520

tggccccgct?gactgtcaac?actgaaggtg?ccccacagat?caaggtttac?gccaacatcg 2580

cccctactaa?cgtgcacgtc?gcgggtgagc?tcccttctaa?ggaagggatc?ttccccgtgg 2640

catgtagcga?cggttacggt?ggcctggtga?ccactgaccc?aaagacggct?gaccccgcct 2700

acgggaaagt?gttcaatcca?cctcgcaaca?tgttgccggg?gcggttcacc?aacttccttg 2760

atgtggctga?ggcgtgtcct?acgtttctgc?attttgaggg?tgacgtaccg?tacgtgacca 2820

caaagacgga?ctcagacagg?gtgctcgccc?agtttgactt?gtctctggca?gcaaaacaca 2880

tgtcaaacac?cttcctggca?ggtctcgccc?agtattacac?acagtacagc?ggcaccatca 2940

acctgcactt?catgttcact?ggacccactg?acgcgaaagc?gcgttacatg?attgcatacg 3000

ccccccctgg?catggagccg?cccaaaacac?ccgaggcggc?cgctcactgc?attcatgcgg 3060

agtgggacac?agggttgaac?tcaaaattca?cattttcaat?cccttacctt?tcggcggctg 3120

actacgcgta?caccgcgtct?gactccgcgg?agaccacaaa?cgtgcaggga?tgggtttgcc 3180

tgtttcaaat?cacacacggg?aaggctgacg?gcgacgcgct?ggtcgttcta?gctagtgccg 3240

gtaaggactt?tgaactgcgt?ttgccagttg?atgctcgcac?gcagaccacc?tctacaggtg 3300

agtcggctga?ccccgtaact?gccaccgttg?agaactacgg?tggtgagaca?caggtccaga 3360

gacgccagca?cacggatgtc?tcgttcatac?tagacagatt?tgtgaaagta?acaccaaaag 3420

accaaatcaa?tgtgttggac?ctgatgcaaa?cccctgcaca?cactttggta?ggcgcgctcc 3480

tccgtactgc?cacttactac?tttgcagatc?tagaagtggc?agtgaaacac?gaggggaacc 3540

ttacctgggt?cccgaatggg?gcgcccgagg?cagcattgga?caacaccacc?aatccaacgg 3600

cctaccacaa?ggcgccgctc?acccggcttg?cactgcctta?cacggcacca?caccgtgtct 3660

tggctactgt?ttacaacggg?aactgtaagt?acggcaagag?ccccgtggcc?aacgcgagag 3720

gtgacctgca?agtgttgacc?ccgaaggcgg?caagaacgct?gcctacctcc?ttcaattacg 3780

gcgccatcaa?agccactcgg?gtgactgaac?tgctttaccg?catgaagagg?gccgaaacgt 3840

actgcccccg?gcctcttttg?gctattcacc?cgagcgaaac?tagacacaaa?caaaagattg 3900

tggcgcctgt?gaagcagctt?ttgaattttg?atctgctcaa?gctggcagga?gacgttgagt 3960

ccaaccctgg?acccttcttc?ttcgctgacg?tcaggtcaaa?tttttccaag?ctggttgaga 4020

ccatcaacca?aatgcaggag?gacatgtcaa?caaaacacgg?acccgacttt?aaccggttgg 4080

tgtctgcgtt?tgaggaactg?gccgctggag?tgagggctat?caggactggt?ctcgacgagg 4140

ccaaaccctg?gtacaagctc?atcaagctac?tgagccgcct?gtcatgcatg?gccgctgtag 4200

cagcacggtc?aaaggaccca?gtccttgtgg?ccatcatgct?ggctgacacc?ggtctcgaga 4260

tcctggacag?tacctttgtc?gtgaagaaga?tctccgactc?gctctccagt?ctctttcacg 4320

tgccggcccc?cgtcttcagt?ttcggagccc?cgattttgtt?ggccgggttg?gtcaaggtcg 4380

cctcgagttt?cttccggtcc?acgcccgaag?accttgagag?agcggagaaa?cagctcaaag 4440

cacgtgacat?caatgacata?ttcgccattc?tcaagaacgg?cgagtggctg?gtcaagctga 4500

tccttgccat?ccgcgactgg?atcaaggcat?ggatcgcctc?agaggaaaag?tttgtcacca 4560

tgacagacct?ggtgcccggt?atccttgaaa?agcagcggga?tctcaacgac?ccaagcaagt 4620

acgaggaggc?caaggagtgg?ctcgacaacg?cgcgccaagc?gtgtttgaag?agcgggaaca 4680

tccacatcgc?aaacctttgc?aaagtggctg?ccccagcacc?cagcaggtcg?aggcccgaac 4740

ccgtggtcgt?ttgccttcgt?ggcaaatcag?gccagggcaa?gagtttcctt?gcgaacgtgc 4800

ttgcacaagc?aatttcaacc?cacttcactg?gcagaaccga?ttcagtttgg?tactgcccac 4860

ctgaccctga?ccacttcgac?ggttacaacc?agcagaccgt?tgtagtaatg?gatgacctgg 4920

gccagaaccc?cgacgggaag?gactttaagt?acttcgccca?aatggtttca?actacggggt 4980

ttatcccgcc?catggcttca?cttgaggaca?aaggcaaacc?tttcaacagc?aaggtcatca 5040

tcgccaccac?caacctgtac?tcgggcttca?ccccgagaac?tatggtgtgc?cctgatgcgc 5100

tgaaccggag?gttccacttt?gacatcgacg?tgagcgctaa?ggacggatac?aaaattaaca 5160

acaaattgga?catcatcaaa?gctcttgaag?acacccacac?caacccagtg?gcaatgtttc 5220

aatacgactg?tgcccttctc?aacggcatgg?ccgttgaaat?gaagagaatg?caacaagaca 5280

tgttcaagcc?tcaaccgccc?ctccagaacg?tctaccagct?tgttcaggag?gtgattgacc 5340

gggtcgagct?ccacgaaaag?gtgtcgaacc?acccgatctt?caagcagatc?tcaattcctt 5400

cccaaaaggc?tgtgctgtac?tttctcattg?agaagggcca?gcacgaagca?gcaattgaat 5460

tctttgaggg?gatggtgtgt?gactccatta?aggaggagct?ccggccccta?atccaacaga 5520

cctcatttgt?gaagcgcgct?tttaagcgcc?tgaaggaaaa?ctttgagatt?gtcgctctgt 5580

gtttgaccct?tttggcgaac?atagtgatca?tgatccgcgg?gactcgcaag?agacagcaga 5640

tagtggacga?tgtagtggac?gagtacactg?agaaggcaaa?catcgccacg?gatgacaaga 5700

ctcttgacga?ggcggaaaag?aaccctctgg?aggccagtgg?tgccaccact?gttggtttca 5760

gagagaaaac?tctcccggga?cacaaggcgg?gtgatgacgt?gagctctgag?cccgccgaac 5820

ccgtggaagg?gcaaccacag?gctgaaggac?cctacaccgg?tccactcgag?cgtcaaaaac 5880

ctctgaaagt?gagagccagg?ctcccacagc?aggaggggcc?ctacgctggc?ccgatggaga 5940

gacagaaacc?gctgaaagtg?aaagtgaaag?ccccggtcgt?taaggaagga?ccttacgaag 6000

gaccggtgaa?gaaacctgtc?gctctgaaag?tgaaagcaaa?gaacttgatt?gtcactgaga 6060

gtggtgctcc?cccgactgac?ttgcaaaaga?tggtcatggg?caacaccaag?cctgttgagc 6120

tcatcctcga?cgggaagacg?gtggccatct?gctgcgccac?cggagtgttt?ggtaccgcct 6180

accttgttcc?tcgccatctt?ttcgcagaga?agtacgacaa?gatcatgttg?gacggcagag 6240

ccatgacaga?cagtgactac?agagtgtttg?agtttgagat?taaagtgaaa?gggcaggaca 6300

tgctctcgga?cgccgcgctc?atggtgctcc?accgtgggaa?tcgcgtgcgg?gacatcacga 6360

agcacttccg?tgatgtggca?agaatgaaga?aaggcacccc?cgtcgtcggc?gtggtcaaca 6420

acgctgatgt?tgggagactg?atcttctctg?gtgaggccct?tacctacaag?gacattgtag 6480

tgtgcatgga?cggagacacc?atgcccggtc?tcttcgccta?caaagccgcc?accaaggcgg 6540

gttactgtgg?aggagccgtt?cttgcaaagg?acggagccga?gactttcatc?gtcggcactc 6600

actccgcagg?cggcaacgga?gttggctact?gctcgtgcgt?ttccaggtct?atgctgctaa 6660

aaatgaaggc?acacatcgat?cccgaaccac?accacgaggg?attgatagtt?gacaccagag 6720

atgttgagga?gcgcgtacat?gtcatgcgca?aaaccaagct?cgcacccacc?gtggcatacg 6780

gtgtattcaa?ccccgaattt?gggcctgccg?ccttgtccaa?ccaggacccg?cgcctgaatg 6840

aaggggttgt?cctcgatgaa?gttatcttct?ctaaacacaa?ggaaaacaca?aagatgtctg 6900

aggaggacaa?agcgctgttc?cgccgctgtg?ctgctgacta?cgcgtcccgc?ctgcacagcg 6960

tgctgggtac?ggcaaacgcc?ccactgagca?tttacgaggc?aattaagggt?gtcgacggac 7020

ttgacgccat?ggaaccagac?accgcgcctg?gcctcccctg?ggccctccag?gggaaacgcc 7080

gtggtgcgct?cattgacttc?gagaacggca?ctgtcggacc?cgaggttgag?gctgctttga 7140

agctcatgga?gaaaagagag?tacaagtttg?tatgccagac?ctttctgaag?gacgagatcc 7200

gtccgatgga?aaaggtacgt?gccggtaaga?ctcgcattgt?cgacgtcctg?cctgttgaac 7260

acattcttta?caccaggatg?atgattggta?gattttgcgc?tcaaatgcac?tcaaacaacg 7320

gaccgcaaat?tggttcggcg?gttggttgta?atcctgatgt?tgattggcaa?agatttggca 7380

cgcactttgc?tcagtacaaa?aacgtgtggg?atgtggacta?ttcggccttt?gacgccaacc 7440

actgtagtga?tgcaatgaac?atcatgtttg?aggaggtgtt?caacacggat?ttcggtttcc 7500

acccaaacgc?tgagtggatc?ttgaaaactc?tcgtggacac?tgaacacgcc?tatgagaaca 7560

aacgcatcac?tgttgaaggc?gggatgccgt?ctggttgttc?cgcgacaagc?atcatcaaca 7620

caattttgaa?caacatctac?gtgctctacg?ccttgcgcag?acactatgag?ggagttgagc 7680

tggactctta?caccatgatc?tcctacggag?acgacatcgt?ggttgcaagt?gatcacgatc 7740

tggactttga?ggccctcaag?cctcacttca?aatcccttgg?tcaaaccatc?actccagctg 7800

acaaaagtga?caaaggtttt?gttcttggtc?actccattac?cgatgtcact?ttcctcaaaa 7860

gacacttcca?catggactat?ggaactgggt?tttacaaacc?tgtgatggct?tcgaagaccc 7920

tcgaggctat?cctctccttt?gcacgtcgtg?ggaccataca?ggagaagttg?atctccgtgg 7980

caggactcgc?cgtccactct?ggacctgatg?agtaccggcg?tctctttgag?cctttccagg 8040

gcctcttcga?gattccaagc?tacagatcac?tttacctgcg?ttgggtgaac?gccgtgtgcg 8100

gtgacgcata?atccctcaga?tgtcacaact?ggcagaaaga?cgttgaggcg?agcgacgccg 8160

taggagtgaa?aagtccgaaa?gggcttttcc?cgcttcctat?ttcaaaaaaa?aaaaaaaaaa 8220

aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aa 8262

<210>2

<211>2332

<212>PRT

<213>Hand-foot-and-mouth?disease?virus

<400>2

Met?Ser?Thr?Thr?Asp?Cys?Phe?Ile?Ala?Leu?Leu?Tyr?Ala?Phe?Arg?Glu

1 5 10 15

Ile?Lys?Thr?Leu?Phe?Leu?Ser?Arg?Thr?Arg?Gly?Lys?Met?Glu?Phe?Thr

20 25 30

Leu?His?Asn?Gly?Glu?Lys?Lys?Thr?Phe?Tyr?Ser?Arg?Pro?Asn?Asn?His

35 40 45

Asp?Asn?Cys?Trp?Leu?Asn?Ala?Ile?Leu?Gln?Leu?Phe?Arg?Tyr?Val?Asp

50 55 60

Glu?Pro?Phe?Phe?Asp?Trp?Val?Tyr?Cys?Ser?His?Glu?Asn?Leu?Thr?Leu

65 70 75 80

Asn?Ala?Ile?Lys?Gln?Leu?Glu?Glu?Ile?Thr?Gly?Leu?Glu?Leu?His?Glu

85 90 95

Gly?Gly?Pro?Pro?Ala?Leu?Val?Ile?Trp?Asn?Ile?Lys?His?Leu?Leu?Asn

100 105 110

Thr?Gly?Ile?Gly?Thr?Ala?Ser?Arg?Pro?Ser?Glu?Val?Cys?Met?Val?Asp

115 120 125

Gly?Thr?Asp?Met?Cys?Leu?Ala?Asp?Phe?His?Ala?Gly?Ile?Phe?Leu?Gln

130 135 140

Gly?Gln?Glu?His?Ala?Val?Phe?Ala?Cys?Val?Thr?Ser?Asn?Gly?Trp?Tyr

145 150 155 160

Ala?Ile?Asp?Asp?Glu?Asp?Phe?Tyr?Pro?Trp?Thr?Pro?Asp?Pro?Ser?Asp

165 170 175

Val?Leu?Val?Phe?Val?Pro?Tyr?Asp?Gln?Glu?Pro?Leu?Asn?Gly?Glu?Trp

180 185 190

Lys?Thr?Lys?Val?Gln?Lys?Arg?Leu?Arg?Gly?Ala?Gly?Gln?Ser?Ser?Pro

195 200 205

Ala?Thr?Gly?Ser?Gln?Asn?Gln?Ser?Gly?Asn?Thr?Gly?Ser?Ile?Ile?Asn

210 215 220

Asn?Tyr?Tyr?Met?Gln?Gln?Tyr?Gln?Asn?Ser?Met?Asp?Thr?Gln?Leu?Gly

225 230 235 240

Asp?Asn?Ala?Ile?Ser?Gly?Gly?Ser?Asn?Glu?Gly?Ser?Thr?Asp?Thr?Thr

245 250 255

Ser?Thr?His?Thr?Thr?Asn?Thr?Gln?Asn?Asn?Asp?Trp?Phe?Ser?Lys?Leu

260 265 270

Ala?Ser?Ser?Ala?Phe?Ser?Gly?Leu?Phe?Gly?Ala?Leu?Leu?Ala?Asp?Lys

275 280 285

Lys?Thr?Glu?Glu?Thr?Thr?Leu?Leu?Glu?Asp?Arg?Ile?Leu?Thr?Thr?Arg

290 295 300

Asn?Gly?His?Thr?Thr?Ser?Thr?Thr?Gln?Ser?Ser?Val?Gly?Val?Thr?Tyr

305 310 315 320

Gly?Tyr?Ala?Thr?Ala?Glu?Asp?Phe?Val?Ser?Gly?Pro?Asn?Thr?Ser?Gly

325 330 335

Leu?Glu?Thr?Arg?Val?Val?Gln?Ala?Glu?Arg?Phe?Phe?Lys?Thr?His?Leu

340 345 350

Phe?Asp?Trp?Val?Thr?Ser?Asp?Pro?Phe?Gly?Arg?Cys?Tyr?Leu?Leu?Glu

355 360 365

Leu?Pro?Thr?Asp?His?Lys?Gly?Val?Tyr?Gly?Ser?Leu?Thr?Asp?Ser?Tyr

370 375 380

Ala?Tyr?Met?Arg?Asn?Gly?Trp?Asp?Val?Glu?Val?Thr?Ala?Val?Gly?Asn

385 390 395 400

Gln?Phe?Asn?Gly?Gly?Cys?Leu?Leu?Val?Ala?Met?Val?Pro?Glu?Leu?Cys

405 410 415

Ser?Ile?Asp?Lys?Arg?Gly?Leu?Tyr?Gln?Leu?Thr?Leu?Phe?Pro?His?Gln

420 425 430

Phe?Ile?Asn?Pro?Arg?Thr?Asn?Met?Thr?Ala?His?Ile?Thr?Val?Pro?Phe

435 440 445

Val?Gly?Val?Asn?Arg?Tyr?Asp?Gln?Tyr?Lys?Val?His?Arg?Pro?Trp?Thr

450 455 460

Leu?Val?Val?Met?Val?Val?Ala?Pro?Leu?Thr?Val?Asn?Thr?Glu?Gly?Ala

465 470 475 480

Pro?Gln?Ile?Lys?Val?Tyr?Ala?Asn?Ile?Ala?Pro?Thr?Asn?Val?His?Val

485 490 495

Ala?Gly?Glu?Leu?Pro?Ser?Lys?Glu?Gly?Ile?Phe?Pro?Val?Ala?Cys?Ser

500 505 510

Asp?Gly?Tyr?Gly?Gly?Leu?Val?Thr?Thr?Asp?Pro?Lys?Thr?Ala?Asp?Pro

515 520 525

Ala?Tyr?Gly?Lys?Val?Phe?Asn?Pro?Pro?Arg?Asn?Met?Leu?Pro?Gly?Arg

530 535 540

Phe?Thr?Asn?Phe?Leu?Asp?Val?Ala?Glu?Ala?Cys?Pro?Thr?Phe?Leu?His

545 550 555 560

Phe?Glu?Gly?Asp?Val?Pro?Tyr?Val?Thr?Thr?Lys?Thr?Asp?Ser?Asp?Arg

565 570 575

Val?Leu?Ala?Gln?Phe?Asp?Leu?Ser?Leu?Ala?Ala?Lys?His?Met?Ser?Asn

580 585 590

Thr?Phe?Leu?Ala?Gly?Leu?Ala?Gln?Tyr?Tyr?Thr?Gln?Tyr?Ser?Gly?Thr

595 600 605

Ile?Asn?Leu?His?Phe?Met?Phe?Thr?Gly?Pro?Thr?Asp?Ala?Lys?Ala?Arg

610 615 620

Tyr?Met?Ile?Ala?Tyr?Ala?Pro?Pro?Gly?Met?Glu?Pro?Pro?Lys?Thr?Pro

625 630 635 640

Glu?Ala?Ala?Ala?His?Cys?Ile?His?Ala?Glu?Trp?Asp?Thr?Gly?Leu?Asn

645 650 655

Ser?Lys?Phe?Thr?Phe?Ser?Ile?Pro?Tyr?Leu?Ser?Ala?Ala?Asp?Tyr?Ala

660 665 670

Tyr?Thr?Ala?Ser?Asp?Ser?Ala?Glu?Thr?Thr?Asn?Val?Gln?Gly?Trp?Val

675 680 685

Cys?Leu?Phe?Gln?Ile?Thr?His?Gly?Lys?Ala?Asp?Gly?Asp?Ala?Leu?Val

690 695 700

Val?Leu?Ala?Ser?Ala?Gly?Lys?Asp?Phe?Glu?Leu?Arg?Leu?Pro?Val?Asp

705 710 715 720

Ala?Arg?Thr?Gln?Thr?Thr?Ser?Thr?Gly?Glu?Ser?Ala?Asp?Pro?Val?Thr

725 730 735

Ala?Thr?Val?Glu?Asn?Tyr?Gly?Gly?Glu?Thr?Gln?Val?Gln?Arg?Arg?Gln

740 745 750

His?Thr?Asp?Val?Ser?Phe?Ile?Leu?Asp?Arg?Phe?Val?Lys?Val?Thr?Pro

755 760 765

Lys?Asp?Gln?Ile?Asn?Val?Leu?Asp?Leu?Met?Gln?Thr?Pro?Ala?His?Thr

770 775 780

Leu?Val?Gly?Ala?Leu?Leu?Arg?Thr?Ala?Thr?Tyr?Tyr?Phe?Ala?Asp?Leu

785 790 795 800

Glu?Val?Ala?Val?Lys?His?Glu?Gly?Asn?Leu?Thr?Trp?Val?Pro?Asn?Gly

805 810 815

Ala?Pro?Glu?Ala?Ala?Leu?Asp?Asn?Thr?Thr?Asn?Pro?Thr?Ala?Tyr?His

820 825 830

Lys?Ala?Pro?Leu?Thr?Arg?Leu?Ala?Leu?Pro?Tyr?Thr?Ala?Pro?His?Arg

835 840 845

Val?Leu?Ala?Thr?Val?Tyr?Asn?Gly?Asn?Cys?Lys?Tyr?Gly?Lys?Ser?Pro

850 855 860

Val?Ala?Asn?Ala?Arg?Gly?Asp?Leu?Gln?Val?Leu?Thr?Pro?Lys?Ala?Ala

865 870 875 880

Arg?Thr?Leu?Pro?Thr?Ser?Phe?Asn?Tyr?Gly?Ala?Ile?Lys?Ala?Thr?Arg

885 890 895

Val?Thr?Glu?Leu?Leu?Tyr?Arg?Met?Lys?Arg?Ala?Glu?Thr?Tyr?Cys?Pro

900 905 910

Arg?Pro?Leu?Leu?Ala?Ile?His?Pro?Ser?Glu?Thr?Arg?His?Lys?Gln?Lys

915 920 925

Ile?Val?Ala?Pro?Val?Lys?Gln?Leu?Leu?Asn?Phe?Asp?Leu?Leu?Lys?Leu

930 935 940

Ala?Gly?Asp?Val?Glu?Ser?Asn?Pro?Gly?Pro?Phe?Phe?Phe?Ala?Asp?Val

945 950 955 960

Arg?Ser?Asn?Phe?Ser?Lys?Leu?Val?Glu?Thr?Ile?Asn?Gln?Met?Gln?Glu

965 970 975

Asp?Met?Ser?Thr?Lys?His?Gly?Pro?Asp?Phe?Asn?Arg?Leu?Val?Ser?Ala

980 985 990

Phe?Glu?Glu?Leu?Ala?Ala?Gly?Val?Arg?Ala?Ile?Arg?Thr?Gly?Leu?Asp

995 1000 1005

Glu?Ala?Lys?Pro?Trp?Tyr?Lys?Leu?Ile?Lys?Leu?Leu?Ser?Arg?Leu

1010 1015 1020

Ser?Cys?Met?Ala?Ala?Val?Ala?Ala?Arg?Ser?Lys?Asp?Pro?Val?Leu

1025 1030 1035

Val?Ala?Ile?Met?Leu?Ala?Asp?Thr?Gly?Leu?Glu?Ile?Leu?Asp?Ser

1040 1045 1050

Thr?Phe?Val?Val?Lys?Lys?Ile?Ser?Asp?Ser?Leu?Ser?Ser?Leu?Phe

1055 1060 1065

His?Val?Pro?Ala?Pro?Val?Phe?Ser?Phe?Gly?Ala?Pro?Ile?Leu?Leu

1070 1075 1080

Ala?Gly?Leu?Val?Lys?Val?Ala?Ser?Ser?Phe?Phe?Arg?Ser?Thr?Pro

1085 1090 1095

Glu?Asp?Leu?Glu?Arg?Ala?Glu?Lys?Gln?Leu?Lys?Ala?Arg?Asp?Ile

1100 1105 1110

Asn?Asp?Ile?Phe?Ala?Ile?Leu?Lys?Asn?Gly?Glu?Trp?Leu?Val?Lys

1115 1120 1125

Leu?Ile?Leu?Ala?Ile?Arg?Asp?Trp?Ile?Lys?Ala?Trp?Ile?Ala?Ser

1130 1135 1140

Glu?Glu?Lys?Phe?Val?Thr?Met?Thr?Asp?Leu?Val?Pro?Gly?Ile?Leu

1145 1150 1155

Glu?Lys?Gln?Arg?Asp?Leu?Asn?Asp?Pro?Ser?Lys?Tyr?Glu?Glu?Ala

1160 1165 1170

Lys?Glu?Trp?Leu?Asp?Asn?Ala?Arg?Gln?Ala?Cys?Leu?Lys?Ser?Gly

1175 1180 1185

Asn?Ile?His?Ile?Ala?Asn?Leu?Cys?Lys?Val?Ala?Ala?Pro?Ala?Pro

1190 1195 1200

Ser?Arg?Ser?Arg?Pro?Glu?Pro?Val?Val?Val?Cys?Leu?Arg?Gly?Lys

1205 1210 1215

Ser?Gly?Gln?Gly?Lys?Ser?Phe?Leu?Ala?Asn?Val?Leu?Ala?Gln?Ala

1220 1225 1230

Ile?Ser?Thr?His?Phe?Thr?Gly?Arg?Thr?Asp?Ser?Val?Trp?Tyr?Cys

1235 1240 1245

Pro?Pro?Asp?Pro?Asp?His?Phe?Asp?Gly?Tyr?Asn?Gln?Gln?Thr?Val

1250 1255 1260

Val?Val?Met?Asp?Asp?Leu?Gly?Gln?Asn?Pro?Asp?Gly?Lys?Asp?Phe

1265 1270 1275

Lys?Tyr?Phe?Ala?Gln?Met?Val?Ser?Thr?Thr?Gly?Phe?Ile?Pro?Pro

1280 1285 1290

Met?Ala?Ser?Leu?Glu?Asp?Lys?Gly?Lys?Pro?Phe?Asn?Ser?Lys?Val

1295 1300 1305

Ile?Ile?Ala?Thr?Thr?Asn?Leu?Tyr?Ser?Gly?Phe?Thr?Pro?Arg?Thr

1310 1315 1320

Met?Val?Cys?Pro?Asp?Ala?Leu?Asn?Arg?Arg?Phe?His?Phe?Asp?Ile

1325 1330 1335

Asp?Val?Ser?Ala?Lys?Asp?Gly?Tyr?Lys?Ile?Asn?Asn?Lys?Leu?Asp

1340 1345 1350

Ile?Ile?Lys?Ala?Leu?Glu?Asp?Thr?His?Thr?Asn?Pro?Val?Ala?Met

1355 1360 1365

Phe?Gln?Tyr?Asp?Cys?Ala?Leu?Leu?Asn?Gly?Met?Ala?Val?Glu?Met

1370 1375 1380

Lys?Arg?Met?Gln?Gln?Asp?Met?Phe?Lys?Pro?Gln?Pro?Pro?Leu?Gln

1385 1390 1395

Asn?Val?Tyr?Gln?Leu?Val?Gln?Glu?Val?Ile?Asp?Arg?Val?Glu?Leu

1400 1405 1410

His?Glu?Lys?Val?Ser?Asn?His?Pro?Ile?Phe?Lys?Gln?Ile?Ser?Ile

1415 1420 1425

Pro?Ser?Gln?Lys?Ala?Val?Leu?Tyr?Phe?Leu?Ile?Glu?Lys?Gly?Gln

1430 1435 1440

His?Glu?Ala?Ala?Ile?Glu?Phe?Phe?Glu?Gly?Met?Val?Cys?Asp?Ser

1445 1450 1455

Ile?Lys?Glu?Glu?Leu?Arg?Pro?Leu?Ile?Gln?Gln?Thr?Ser?Phe?Val

1460 1465 1470

Lys?Arg?Ala?Phe?Lys?Arg?Leu?Lys?Glu?Asn?Phe?Glu?Ile?Val?Ala

1475 1480 1485

Leu?Cys?Leu?Thr?Leu?Leu?Ala?Asn?Ile?Val?Ile?Met?Ile?Arg?Gly

1490 1495 1500

Thr?Arg?Lys?Arg?Gln?Gln?Ile?Val?Asp?Asp?Val?Val?Asp?Glu?Tyr

1505 1510 1515

Thr?Glu?Lys?Ala?Asn?Ile?Ala?Thr?Asp?Asp?Lys?Thr?Leu?Asp?Glu

1520 1525 1530

Ala?Glu?Lys?Asn?Pro?Leu?Glu?Ala?Ser?Gly?Ala?Thr?Thr?Val?Gly

1535 1540 1545

Phe?Arg?Glu?Lys?Thr?Leu?Pro?Gly?His?Lys?Ala?Gly?Asp?Asp?Val

1550 1555 1560

Ser?Ser?Glu?Pro?Ala?Glu?Pro?Val?Glu?Gly?Gln?Pro?Gln?Ala?Glu

1565 1570 1575

Gly?Pro?Tyr?Thr?Gly?Pro?Leu?Glu?Arg?Gln?Lys?Pro?Leu?Lys?Val

1580 1585 1590

Arg?Ala?Arg?Leu?Pro?Gln?Gln?Glu?Gly?Pro?Tyr?Ala?Gly?Pro?Met

1595 1600 1605

Glu?Arg?Gln?Lys?Pro?Leu?Lys?Val?Lys?Val?Lys?Ala?Pro?Val?Val

1610 1615 1620

Lys?Glu?Gly?Pro?Tyr?Glu?Gly?Pro?Val?Lys?Lys?Pro?Val?Ala?Leu

1625 1630 1635

Lys?Val?Lys?Ala?Lys?Asn?Leu?Ile?Val?Thr?Glu?Ser?Gly?Ala?Pro

1640 1645 1650

Pro?Thr?Asp?Leu?Gln?Lys?Met?Val?Met?Gly?Asn?Thr?Lys?Pro?Val

1655 1660 1665

Glu?Leu?Ile?Leu?Asp?Gly?Lys?Thr?Val?Ala?Ile?Cys?Cys?Ala?Thr

1670 1675 1680

Gly?Val?Phe?Gly?Thr?Ala?Tyr?Leu?Val?Pro?Arg?His?Leu?Phe?Ala

1685 1690 1695

Glu?Lys?Tyr?Asp?Lys?Ile?Met?Leu?Asp?Gly?Arg?Ala?Met?Thr?Asp

1700 1705 1710

Ser?Asp?Tyr?Arg?Val?Phe?Glu?Phe?Glu?Ile?Lys?Val?Lys?Gly?Gln

1715 1720 1725

Asp?Met?Leu?Ser?Asp?Ala?Ala?Leu?Met?Val?Leu?His?Arg?Gly?Asn

1730 1735 1740

Arg?Val?Arg?Asp?Ile?Thr?Lys?His?Phe?Arg?Asp?Val?Ala?Arg?Met

1745 1750 1755

Lys?Lys?Gly?Thr?Pro?Val?Val?Gly?Val?Val?Asn?Asn?Ala?Asp?Val

1760 1765 1770

Gly?Arg?Leu?Ile?Phe?Ser?Gly?Glu?Ala?Leu?Thr?Tyr?Lys?Asp?Ile

1775 1780 1785

Val?Val?Cys?Met?Asp?Gly?Asp?Thr?Met?Pro?Gly?Leu?Phe?Ala?Tyr

1790 1795 1800

Lys?Ala?Ala?Thr?Lys?Ala?Gly?Tyr?Cys?Gly?Gly?Ala?Val?Leu?Ala

1805 1810 1815

Lys?Asp?Gly?Ala?Glu?Thr?Phe?Ile?Val?Gly?Thr?His?Ser?Ala?Gly

1820 1825 1830

Gly?Asn?Gly?Val?Gly?Tyr?Cys?Ser?Cys?Val?Ser?Arg?Ser?Met?Leu

1835 1840 1845

Leu?Lys?Met?Lys?Ala?His?Ile?Asp?Pro?Glu?Pro?His?His?Glu?Gly

1850 1855 1860

Leu?Ile?Val?Asp?Thr?Arg?Asp?Val?Glu?Glu?Arg?Val?His?Val?Met

1865 1870 1875

Arg?Lys?Thr?Lys?Leu?Ala?Pro?Thr?Val?Ala?Tyr?Gly?Val?Phe?Asn

1880 1885 1890

Pro?Glu?Phe?Gly?Pro?Ala?Ala?Leu?Ser?Asn?Gln?Asp?Pro?Arg?Leu

1895 1900 1905

Asn?Glu?Gly?Val?Val?Leu?Asp?Glu?Val?Ile?Phe?Ser?Lys?His?Lys

1910 1915 1920

Glu?Asn?Thr?Lys?Met?Ser?Glu?Glu?Asp?Lys?Ala?Leu?Phe?Arg?Arg

1925 1930 1935

Cys?Ala?Ala?Asp?Tyr?Ala?Ser?Arg?Leu?His?Ser?Val?Leu?Gly?Thr

1940 1945 1950

Ala?Asn?Ala?Pro?Leu?Ser?Ile?Tyr?Glu?Ala?Ile?Lys?Gly?Val?Asp

1955 1960 1965

Gly?Leu?Asp?Ala?Met?Glu?Pro?Asp?Thr?Ala?Pro?Gly?Leu?Pro?Trp

1970 1975 1980

Ala?Leu?Gln?Gly?Lys?Arg?Arg?Gly?Ala?Leu?Ile?Asp?Phe?Glu?Asn

1985 1990 1995

Gly?Thr?Val?Gly?Pro?Glu?Val?Glu?Ala?Ala?Leu?Lys?Leu?Met?Glu

2000 2005 2010

Lys?Arg?Glu?Tyr?Lys?Phe?Val?Cys?Gln?Thr?Phe?Leu?Lys?Asp?Glu

2015 2020 2025

Ile?Arg?Pro?Met?Glu?Lys?Val?Arg?Ala?Gly?Lys?Thr?Arg?Ile?Val

2030 2035 2040

Asp?Val?Leu?Pro?Val?Glu?His?Ile?Leu?Tyr?Thr?Arg?Met?Met?Ile

2045 2050 2055

Gly?Arg?Phe?Cys?Ala?Gln?Met?His?Ser?Asn?Asn?Gly?Pro?Gln?Ile

2060 2065 2070

Gly?Ser?Ala?Val?Gly?Cys?Asn?Pro?Asp?Val?Asp?Trp?Gln?Arg?Phe

2075 2080 2085

Gly?Thr?His?Phe?Ala?Gln?Tyr?Lys?Asn?Val?Trp?Asp?Val?Asp?Tyr

2090 2095 2100

Ser?Ala?Phe?Asp?Ala?Asn?His?Cys?Ser?Asp?Ala?Met?Asn?Ile?Met

2105 2110 2115

Phe?Glu?Glu?Val?Phe?Asn?Thr?Asp?Phe?Gly?Phe?His?Pro?Asn?Ala

2120 2125 2130

Glu?Trp?Ile?Leu?Lys?Thr?Leu?Val?Asp?Thr?Glu?His?Ala?Tyr?Glu

2135 2140 2145

Asn?Lys?Arg?Ile?Thr?Val?Glu?Gly?Gly?Met?Pro?Ser?Gly?Cys?Ser

2150 2155 2160

Ala?Thr?Ser?Ile?Ile?Asn?Thr?Ile?Leu?Asn?Asn?Ile?Tyr?Val?Leu

2165 2170 2175

Tyr?Ala?Leu?Arg?Arg?His?Tyr?Glu?Gly?Val?Glu?Leu?Asp?Ser?Tyr

2180 2185 2190

Thr?Met?Ile?Ser?Tyr?Gly?Asp?Asp?Ile?Val?Val?Ala?Ser?Asp?His

2195 2200 2205

Asp?Leu?Asp?Phe?Glu?Ala?Leu?Lys?Pro?His?Phe?Lys?Ser?Leu?Gly

2210 2215 2220

Gln?Thr?Ile?Thr?Pro?Ala?Asp?Lys?Ser?Asp?Lys?Gly?Phe?Val?Leu

2225 2230 2235

Gly?His?Ser?Ile?Thr?Asp?Val?Thr?Phe?Leu?Lys?Arg?His?Phe?His

2240 2245 2250

Met?Asp?Tyr?Gly?Thr?Gly?Phe?Tyr?Lys?Pro?Val?Met?Ala?Ser?Lys

2255 2260 2265

Thr?Leu?Glu?Ala?Ile?Leu?Ser?Phe?Ala?Arg?Arg?Gly?Thr?Ile?Gln

2270 2275 2280

Glu?Lys?Leu?Ile?Ser?Val?Ala?Gly?Leu?Ala?Val?His?Ser?Gly?Pro

2285 2290 2295

Asp?Glu?Tyr?Arg?Arg?Leu?Phe?Glu?Pro?Phe?Gln?Gly?Leu?Phe?Glu

2300 2305 2310

Ile?Pro?Ser?Tyr?Arg?Ser?Leu?Tyr?Leu?Arg?Trp?Val?Asn?Ala?Val

2315 2320 2325

Cys?Gly?Asp?Ala

2330

Claims

1.O the full genome of type foot and mouth disease virus variant is characterized in that: its nucleotides sequence is classified as shown in the SEQ IDNO:1.

2. the infective cloned plasmids of the described O type of claim 1 foot and mouth disease virus variant, its microbial preservation number are: CGMCC No.3719.

3. by the protein of the described genes encoding of claim 1, it is characterized in that: its aminoacid sequence is shown in the SEQID NO:2.

4. contain the described expression carrier of claim 1.

5. the host cell that contains the described expression vector of claim 4.

6. the described proteinic advantage neutralizing epitope of claim 3 is characterized in that its aminoacid sequence is: ¹ ⁴⁷DLQVLTPK ¹⁵⁴, wherein, the key amino acid residue of this epi-position is 147 and 153 amino acids residues.

7. the described proteinic advantage neutralizing epitope of claim 3 is characterized in that its aminoacid sequence is: ¹ ⁴⁵RGDLQVLTPK ¹⁵⁴, wherein, the key amino acid residue of this epi-position is 147,149 and 150 amino acids residues.

8. the purposes of the described gene of claim 1 in preparation prevention or treatment O type foot and mouth disease virus vaccine.

9. the purposes of the described protein of claim 3 in preparation prevention or treatment O type foot and mouth disease virus vaccine.

10. claim 6 or the 7 described advantage neutralizing epitopes purposes in preparation prevention or treatment O type foot and mouth disease virus vaccine.