CN103849637B - The acidproof mutant strain of O type foot and mouth disease virus, its capsid protein carried and encoding gene thereof and application - Google Patents

Abstract

The invention discloses the acid-resistant mutant strain of O-type foot-and-mouth disease virus, the capsid protein carried by it and its coding gene, and the invention also discloses the key amino acid site and application for determining the acid-resistant characteristic of O-type foot-and-mouth disease virus. In the present invention, the parent strain of O-type foot-and-mouth disease virus is screened by continuous passage under acid pressure, and a mutant strain with strong acid resistance is obtained. The analysis finds that the key amino acid determining the acid-resistant property of O-type foot-and-mouth disease virus is VP1? N17D mutation, the foot-and-mouth disease virus that only carries the mutation of this site has similar acid resistance to the mutant strain, and has good replication ability and immunogenicity. The acid-resistant determinant site found in the invention can be applied to the acid-resistant transformation of the foot-and-mouth disease vaccine seed virus and the development of a new generation of foot-and-mouth disease genetic engineering vaccine. The acid-resistant mutant strain rN17D of the invention can be used as a high-quality inactivated vaccine to produce seed virus, can increase the content of 146S effective antigen in the inactivated vaccine, and increase the immune efficacy of the vaccine.

Description

O-type foot-and-mouth disease virus acid-resistant mutant strain, its capsid protein and its encoding gene and application

technical field

The invention relates to an acid-resistant mutant strain of foot-and-mouth disease virus and the capsid protein and coding gene carried by it, in particular to an infectious cDNA plasmid clone of an acid-resistant mutant strain of an O-type foot-and-mouth disease virus and rescue of the infectious cDNA plasmid clone of an acid-resistant mutant strain of an O-type foot-and-mouth disease virus The acid-resistant mutant strain of O-type foot-and-mouth disease virus, and the invention also relates to the VP1N17D mutation site and application for determining the acid-resistant property of the O-type foot-and-mouth disease virus acid-resistant strain, belonging to the field of foot-and-mouth disease virus prevention and control.

Background technique

Foot-and-mouth disease (Foot and Mouth Disease, FMD) is caused by Foot and Mouth Disease Virus (Foot and Mouth Disease Virus, FMDV), an acute, febrile, highly contagious infectious disease that mainly affects cloven-hoofed animals. Once the disease breaks out, it will have a serious impact on the social economy, so it is called a political and economic disease in the world, and has always been highly valued by governments.

FMDV is a member of the foot-and-mouth disease virus genus of the Picornaviridae family and belongs to single-stranded positive-sense RNA viruses. FMDV is highly sensitive to acid, the optimum pH value is 7.4-7.6, and the capsid will depolymerize when the pH value is slightly lower than neutral conditions. The acid sensitivity of FMDV is necessary for the cleavage of the viral capsid. After the virus enters the cell, the acidic environment of the endosome induces the capsid to disintegrate, so that the RNA genome is released in the infected cell (Carrillo et al., 1985). Although the phenomenon that FMDV uses endosome acidification to uncoat has been discovered very early, the molecular mechanism of acid-mediated FMDV uncoating is still unclear.

The acid sensitivity of foot-and-mouth disease virus has always been a technical problem in the production of inactivated vaccines. For FMD vaccines, 146S virion was the most effective antigen for inactivated vaccines to induce neutralizing antibodies. However, because FMDV is extremely sensitive to an acidic environment, when FMDV is propagated in vitro, acid production by cell metabolism can easily reduce the pH of the FMDV culture environment. When the pH value is slightly lower than neutral conditions, the capsid of FMDV will be cleaved, resulting in The effective antigenicity of FMDV is reduced, and its immune efficacy of the inactivated vaccine thus prepared will decrease (Doel, T.R., and W.K.Chong.1982.Comparativeimmunogenicityof146S,75Sand12Sparticlesoffoot-and-mouthdiseasevirus.ArchVirol73:185-191.). In addition, the FMDV capsid is also sensitive to temperature, which requires a complete cold chain system during the transportation and storage of FMDV inactivated vaccines, resulting in a substantial increase in costs. Therefore, the development of highly acid-resistant foot-and-mouth disease vaccine strains is of great significance for improving the immune efficacy of inactivated vaccines.

Contents of the invention

One of the objects of the present invention is to construct the infectious cDNA plasmid clone of O-type FMDV acid-resistant mutant strain;

Two of the purpose of the present invention is to provide the O-type foot-and-mouth disease virus acid-resistant mutant virus rescued by the O-type FMDV acid-resistant mutant strain infectious cDNA plasmid clone;

The third object of the present invention is to provide the key amino acid that determines the acid-resistant characteristic of O-type foot-and-mouth disease virus;

The fourth object of the present invention is to apply the infectious cDNA plasmid of the O-type FMDV acid-resistant mutant strain and the rescued acid-resistant mutant strain virus to the field of preventing or treating foot-and-mouth disease.

The fifth object of the present invention is to apply the key amino acid that determines the acid resistance of Type O foot-and-mouth disease virus to the field of prevention or treatment of foot-and-mouth disease.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

An infectious cDNA plasmid clone (pYS-N17D) of the O-type FMDV acid-resistant mutant strain, the full-length nucleotide sequence of the O-type FMDV acid-resistant mutant strain infectious cDNA plasmid clone is shown in SEQ ID No. 1; its microbial deposit number Is: CGMCCNO.6868; storage time: November 22, 2012; the classification of the recombinant plasmid is named: O-type FMDV acid-resistant mutant strain infectious cDNA clone plasmid pYS-N17D; depository unit: China Microorganism Collection Management Committee Common Microorganisms Center; Deposit Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China.

In the present invention, firstly, the O-type FMDVO/YS/CHA/05 strain is continuously passed down for 15 generations under acid pressure, and an O-type FMDV mutant strain with high acid resistance is obtained by screening, which is named O-AR strain. The amino acid sequence of the capsid protein carried by the mutant strain is shown in SEQ ID No.3, and its coding gene is shown in SEQ ID No.2. The invention extracts respectively the virus RNAs of three generations of O-type foot-and-mouth disease virus P5, P10 and P15 obtained by acid pressure screening, and compares the acid resistance of the three generations of viruses. The test results found that the P5, P10 and P15 strains were all acid-resistant strains of O-type FMDV and had similar acid-resistant characteristics, which indicated that the parental strains obtained the acid-resistant phenotype after screening in vitro to the fifth generation, and this acid-resistant phenotype Stable until the 15th generation.

In order to analyze the amino acid sites that determine the acid-resistant characteristics of FMDV, the genome sequences of P5, P10, P15 strains and their parent strains were determined and the sequences of the structural protein coding regions were compared. Sequence comparison analysis found that the structural protein gene of the P5 acid-resistant strain had three point mutations: G218A, A512C, and A1618G. Asparagine (S73N), aspartic acid at position 80 of capsid protein VP2 was mutated to alanine (D86A), and asparagine at position 17 of capsid protein VP1 was mutated to aspartic acid (N17D). P10 and P15 acid-resistant strains also only have these three mutation sites, and no other amino acid mutations have been increased due to the increase of acid stress screening generations. In addition, the 5th-generation acid-resistant strain can also stably exist after 10 generations of continuous passage without acid stress, and the acid-resistant ability of the strain remains unchanged. These results indicated that the above-mentioned three mutated amino acids may be related to the acid-resistant properties of O-type FMDV.

According to the comparison and analysis of the structural gene sequence of acid-resistant strains, the variation of its amino acid residues is known, and the present invention utilizes the site-directed mutagenesis technique to respectively construct the infection of single-site and combined-site mutations corresponding to the three mutation sites of S73N, D86A and VP1N17D. The cloned plasmids pS73N, pD86A, pN17D, pS73N/D86A, pS73N/N17D, pD86A/N17D and pS73N+D86A+N17D were successfully rescued from these 7 mutant strains. After 5 generations of continuous passage on BHK-21 cells, the mutation sites were verified to be correct by whole-genome sequencing, and then the acid resistance of these rescued mutant viruses and their parental viruses were compared and analyzed. The test results found that the virus titers of the virus strains containing the N17D mutation (rN17D, rS73N/N17D, rD86A/N17D and rS73N+D86A+N17D) did not change significantly after being treated for 30 minutes at pH 6.0 and pH 7.4, and these viruses It has similar acid resistance to the P5 strain. The titers of rS73N, rD86A and rS73N/D86A mutants were 10,000 times lower than those treated at pH 7.4 after treatment at pH 6.0 for 30 minutes, which was similar to the titer of the parental virus after treatment at this condition. The above test results show that only the VP1N17D mutation (the 17th amino acid of VP1 protein is mutated from asparagine to aspartic acid) is the molecular factor that determines the acid-resistant phenotype of O-type FMDV, and the amino acid of the mutated VP1 protein (VP1N17D) The sequence is shown in SEQIDNo.5, and its coding gene is shown in SEQIDNo.4.

The FMD mutant strain virus (N17D) carrying only the VP1N17D mutation (VP1N17D) had similar immunogenicity to the parental virus O/YS/CHA/05 after acting in a neutral (pH 7.4) environment, but in acid (pH 6 .0) The immunogenicity of the N17D mutant virus was significantly better than that of the parent virus O/YS/CHA/05 after being treated for 30 minutes in the environment. The above experimental results show that the mutation of the 17th amino acid of the VP1 protein from asparagine to aspartic acid (N17D mutation) not only does not affect the antigenicity of the virus, but endows the virus and its antigen with acid resistance, making it resistant to acid in an acid environment. still have good immunogenicity. In view of the sensitivity of foot-and-mouth disease virus to acid, the capsid of 146S virion will be cracked in a slightly acidic environment, resulting in a decrease in the effective antigenic amount of FMDV and a decrease in the immune efficacy of the prepared inactivated vaccine. The present invention provides a theoretical and experimental basis for the production of O-type foot-and-mouth disease virus inactivated vaccine by preparing the acid-resistant strain of O-type foot-and-mouth disease virus and determining its molecular determinants. The technical problem of acid inactivation lays a material foundation.

Description of drawings

Fig. 1 Comparison of the acid tolerance of strains of different generations of O-type foot-and-mouth disease virus acid stress screening.

Fig. 2 Immunofluorescence detection of BHK-21 cells infected by the O-type foot-and-mouth disease FMDV mutant strain pretreated in an acidic environment at pH 6.0.

Fig. 3 Virus titers of the mutant strain virus in different pH environments for 30 minutes.

Figure 4 Comparison of the replication ability of the mutant strain virus rN17D with that of the parental virus.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Experimental Materials

1. Cells, viruses and plasmids

BHK-21 cell (ATCC Culture Collection Center, ATCCNumber: CCL-10); O-type FMDVO/YS/CHA/05 strain (GenBank accession number: HM008917) and the infectious cDNA clone of the virus can be published according to the literature (Chinese Invention Patent Publication No.: CN101838658A; Invention Name: Type O Foot-and-Mouth Disease Virus Variant Strain and Its Encoding Gene and Application); The pOK-12 vector was donated by Messing (1991) (GenBank accession number of the pOK-12 vector: AF223639).

2. Reagents

PrimeSTAR DNA polymerase, PrimerscriptII reverse transcriptase, and various restriction endonucleases were all purchased from TaKaRa Company. T4 DNA ligase was purchased from NEB Company. SimplyPTotalRNAExtractionkit RNA extraction kit was purchased from BioFlux Company. In vitro transcription was performed using the RiboMAXTM LargeScaleRNAProductionSystems-T7 kit from Promega. The transfection reagent was QIAGEN's TransfectionReagent Transfection Kit. Goat anti-mouse IgG fluorescent antibody was purchased from Sigma.

3. Preparation of PBS with different pH values

The formula of PBS was 50mM NaH ₂ PO ₄ , 100mM NaCl, and the pH values of the buffer were adjusted to 7.4, 6.0 and 5.0 with concentrated hydrochloric acid.

Screening and identification of embodiment 1O type foot-and-mouth disease virus acid-resistant mutant

1. Screening of O-type foot-and-mouth disease virus acid-resistant mutants

Take 10 μL (10 ⁶ TCID ₅₀ /100ul) of the O-type FMDVO/YS/CHA/05 strain and add it to 300 μL PBS (pH6.0) for 1 hour at room temperature, neutralize it with 100 μL 1MTris (pH7.4) and inoculate BHK-21 cells, and wait for 80 After the cells appeared CPE, the cells were repeatedly frozen and thawed three times to collect the virus and measure the virus titer. The virus was used as the seed for the next round of acid stress selection until the 15th generation. Parents were treated in the same way with PBS of pH 7.4, and continuously passed for 15 generations. After 15 passages were screened at pH 6.0, the 5th passage (P5), 10th passage (P10) and 15th passage (P15) viruses were selected to test their tolerance to pH 6.0 and pH 7.4 acidity.

2. Indirect Immunofluorescence Analysis of Acid-resistant Mutants

Take 10 μL of the rescued mutant virus and add it to 300 μL PBS (pH 6.0) for 1 hour at room temperature, then neutralize it with 100 μL Tris-HCL (pH 7.6). BHK-21 cells were inoculated into 96-well plates, and when they grew to 90% monolayer, they were inoculated with the virus treated under acidic conditions at pH 6.0. After 12 hours, the infected BHK-21 cells were fixed with pre-cooled absolute ethanol for 15 minutes, and added O Type FMDV-specific monoclonal antibody 8E8, reacted at 37°C for 1 hour, washed with PBST, added fluorescently labeled goat anti-mouse IgG (Sigma) and reacted at 37°C for 45 minutes, washed with PBST, sealed with 70% glycerol, and observed under a fluorescent microscope.

The experimental results are shown in Figure 1. After treatment at pH 7.4 for 30 minutes, the titers of the parent virus (type O FMDVO/YS/CHA/05 strain), P5, P10 and P15 viruses were all 10 ^5.75 TCID ₅₀ /100uL. Treated at pH6.0 for 30min, the virus titers of P5, P10, and P15 were the same as those at pH7.4, still 10 ^5.75 TCID ₅₀ /100uL, while the parent virus O/YS/CHA/05 strain was treated here The virus titer under the condition is only 10 ^1.75 TCID ₅₀ /100uL, which is 10000 times lower than that under the condition of pH7.4. Indirect immunofluorescence test also further confirmed this result (Figure 2). The above test results show that the P5, P10 and P15 strains are all O-type FMDV acid-resistant strains, and the parental strains obtained the acid-resistant phenotype after screening in vitro to the fifth generation, and this acid-resistant phenotype existed stably until the end of the experiment. 15 generations.

In the present invention, the screened O-type FMDV with high acid resistance is named O-AR strain, the amino acid sequence of the capsid protein carried by the O-AR strain is shown in SEQ ID No.3, and its coding gene is SEQ ID No.2 shown.

Example 2 Construction of infectious cDNA clone plasmid pYS-N17D of O-type FMDV acid-resistant mutant strain and rescue of O-type foot-and-mouth disease virus acid-resistant mutant strain virus and analysis of acid-resistant characteristics

1. Viral RNA extraction, cDNA synthesis and DNA sequence alignment

In order to analyze the amino acid sites that determine the acid-resistant characteristics of FMDV, the genome sequences of P5, P10, P15 strains and their parent strains were determined and the sequences of the structural protein coding regions were compared.

According to the instructions of the SimplyPTotalRNAExtraction kit, the viral RNAs of three passages of O-type foot-and-mouth disease virus P5, P10 and P15 obtained by the acid pressure screening in Example 1 were respectively extracted, and the extracted RNA was dissolved with 26 μL of DEPC water.

Using the extracted viral RNA as a template and O1igo-dT (15T) as a reverse transcription primer, cDNA was synthesized under the action of reverse transcriptase PrimeScript. The reaction conditions are: 25°C for 10 minutes, 42°C for 60 minutes, and 4°C for 20 minutes. The reaction system is 50 μL, including: viral RNA 26 μL; dNTP (25 mM) 6 μL; PrimeScript reverse transcriptase 1 μL; 5×PSRT-Buffer 10 μL; Oligo-dT 6 μL; RNA Inhibitor 1 μL. Using the reverse transcription product as a template, a pair of primers for the O-type FMDV structural protein region was used to amplify the viral structural protein coding region. This pair of primers is pU (5'ACCCCAGCACGGCAACTTTAT3') and pL (5'CCCAGGTCATCCATTACTACAAC3'). The PCR reaction system was 50 μL, including: ddH ₂ O 28 μL; 5×PSbuffer 10 μL; dNTP 4 μL; PU (10 pmol/μL) 2 μL; PL (10 pmol/μL) 2 μL; template 3 μL; PrimeSTAR HS DNA polymerase 1 μL. The reaction conditions of PCR were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 1.5 min, a total of 30 cycles; finally, extension at 72°C for 10 min. The PCR amplification products were recovered and purified by gel and sequenced. The primers used for sequencing were PCR primers PU and PL, p2297L5'CCGTTGAACTGATTCCCCACT3', p2129U5'TGTGCAGGCAGAGCGGTTCTT3', p3575L5'GCCGTTGGATTGGTGGTGTTG3'and p3335U5'TGGTGAGACACAGGTCCAGAG3'. The measured sequence was compared with the corresponding sequence of the parent strain O/YS/CHA/05.

Sequence comparison analysis found that the P5 acid-resistant strain structural protein gene has three point mutations, namely G218A, A512C, and A1618G. These nucleotide mutations lead to three amino acid mutations, which are capsid protein VP4 (GenBank accession number: HM008917) The mutation of the 73rd serine to asparagine (S73N mutation), the 80th aspartic acid mutation of capsid protein VP2 (GenBank accession number: HM008917) to alanine (D86A mutation) and the capsid protein VP1 (GenBank accession number: HM008917) Accession number: HM008917) the 17th asparagine was mutated to aspartic acid (N17D mutation).

P10 and P15 acid-resistant strains also only have these three mutation sites, and no other amino acid mutations have been increased due to the increase of acid stress screening generations. In addition, the 5th-generation acid-resistant strain can also stably exist after 10 generations of continuous passage without acid stress, and the acid-resistant ability of the strain remains unchanged. These results indicated that the above-mentioned three mutated amino acids may be related to the acid-resistant properties of O-type FMDV.

According to the comparison and analysis of the structural gene sequence of acid-resistant strains, the variation of its amino acid residues was known, and the infectious cDNA clone plasmids pS73N, pD86A, pN17D, pS73N/D86A, pS73N/N17D were constructed by site-directed mutagenesis technology, respectively. , pD86A/N17D and pS73N+D86A+N17D, and successfully rescued the seven mutant viruses. It was stable after 5 consecutive passages on BHK-21 cells, and the mutation sites were verified to be correct by whole genome sequencing, and then the acid resistance of these rescued mutant viruses and their parental viruses were compared and analyzed. The results are shown in Figure 3. Virus strains containing N17D mutations (rN17D, rS73N/N17D, rD86A/N17D and rS73N+D86A+N17D) had no significant change in virus titer after treatment at pH 6.0 and pH 7.4 for 30 minutes. And these viruses have similar acid tolerance with P5 strain. The titers of rS73N, rD86A and rS73N/D86A mutants were 10,000 times lower than those treated at pH 7.4 after treatment at pH 6.0 for 30 minutes, which was similar to the titer of the parental virus after treatment at this condition. The above test results showed that only the VP1N17D mutation was the molecular factor determining the acid-resistant phenotype of O-type FMDV.

2. Site-directed mutagenesis

Press Quik Site-DirectedMutagenesisKit说明书，用PrimeSTARDNAPolymerase通过PCR的方法在感染性cDNA克隆上利用引物（S73N-UP-5'ACGACTGGTTTTCAAAGCTAGCCAATTCTGCTTTTAGCGGTCTTTTCGG3'，S73N-Low-5'CCGAAAAGACCGCTAAAAGCAGAATTGGCTAGCTTTGAAAACCAGTCGT3'；D86A-UP-5'TGCTACCTGCTGGAACTCCCAACTGCCCACAAAGGTGTCTACGGTAGCC3'，D86A-Low -5'ACCGTAGACACCTTTGTGGGCAGTTGGGAGTTCCAGCAGGTAGCACCGT3';N17D-UP-5'GTAACTGCCACCGTTGAGGACTACGGTGGTGAGACACAGGTCCAGAG3',N17D-Low-5'ACCTGTGTCTCACCACCGTAGTCCTCAACGGTGGCAGTTACGGGGTCAGC3') introduced point mutations respectively. Single point mutations and combined site mutations were carried out on the infectious cDNA clones, and the obtained clones were named pS73N, pD86A, pN17D, pS73N+D86A, pS73N+N17D, pD86A+N17D, pS73N+D86A+N17D. The PCR program is: 95°C for 5min; 95°C for 30s, 68°C for 9min, 18 cycles; 72°C for 10min. Then the methylated template pOK-C in the PCR product was degraded with DpnI (37°C, 1h), the treated PCR product was transformed into competent cell DH5a, and the positive plasmid was extracted and identified. After being identified correctly by sequencing, it is used to rescue the mutant virus.

3. Rescue of Mutant Virus

Recombinant plasmids pS73N, pD86A, pN17D, pS73N+D86A, pS73N+N17D, pD86A+N17D, pS73N+D86A+N17D were digested and linearized with restriction endonuclease EcoRV, followed by RiboMAXTM LargeScaleRNAProductionSystems-T7 system instructions for extracellular transcription and reaction system It is: 25mmol/LrNTP6μL, 5× buffer 4μL, T7RNA polymerase mixture 2μL, EcoRV linearized and corresponding site-directed mutation pOKT7-O/YS/CHA/05 recombinant plasmid 8μL (2μg), the total volume is 20μL. After the reaction was well mixed, it was incubated at 37°C for 2.5h, digested with RNase-FreeDNase for 15min, the DNA template was removed, and the transcription product was purified by phenol-chloroform extraction. When the BHK-21 cells in the 6-well plate grow to 60%-90% monolayer, wash the cells twice with PBS, and add 1.5 mL of DMEM cell culture medium containing 2% fetal bovine serum. The RNA obtained by extracellular transcription was transfected into BHK-21 cells according to the instruction of QIAGEN's EffecteneR Transfection Reagent Transfection Kit for virus rescue. The transfected cells were cultured at 37°C in 5% CO ₂ , and the cytopathic changes were observed. The virus was harvested in about 3 days, and the BHK-21 cells were subcultured after repeated freezing and thawing 3 times until the virus could produce stable CPE.

4. Determination of Virus Titer

Virus titers were determined by the TCID ₅₀ (tissue half infectious dose) method. First, select BHK-21 cells with good growth status and normal shape. After trypsinization, add 2% DMEM according to the amount of 5-8×10 ⁵ cells per ml of culture medium, blow off the cells with a pipette, and add to In a 96-well plate, 100 μL per well. Viruses were then serially diluted 10-fold in serum-free DMEM. Remove the cultured cells from the 96-well plate and add the above virus dilution, 50 μL per well, and make 4 replicate wells for each dilution. Then, the 96-well plate was cultured in a CO ₂ incubator at 37°C. After 3 days, the cell lesion was observed under a microscope, and the TCID ₅₀ /100 μL was calculated according to the Karber method.

When determining the acid sensitivity of the virus, the virus titer was also determined by TCID50 _by treating the virus with PBS at different pH values before diluting the virus. Add 30 μL of virus liquid to 600 μL of PBS with different pH values (the pH value after adding the virus is the final pH value), and add 120 μL Tris-HCL (pH 7.6) to neutralize after 1 hour, and the virus dilution factor is 25 times at this time; Add 100 μL of the virus solution in this tube to 300 μL of serum-free DMEM culture solution and mix by pipetting up and down. At this time, the virus is diluted 100 times, and then serially diluted 10 times to 10 ^-7 . Virus titers after acid treatment were determined as described above.

The virus titer determination results of the rescued mutant strains are shown in Figure 3. Virus strains containing N17D mutations (rN17D, rS73N/N17D, rD86A/N17D and rS73N+D86A+N17D) were treated at pH 6.0 and pH 7.4 After 30 minutes, the virus titers did not change significantly, and these viruses had similar acid resistance to the P5 strain. The titers of rS73N, rD86A and rS73N/D86A mutants were 10,000 times lower than those treated at pH 7.4 after treatment at pH 6.0 for 30 minutes, which was similar to the titer of the parental virus after treatment at this condition. The above test results showed that only the VP1N17D mutation was the molecular factor determining the acid-resistant phenotype of O-type FMDV.

5. One-step growth curve drawing

In order to analyze whether the VP1N17D mutation affects the growth and replication ability of O-type FMDV, the acid-resistant mutant strain virus rescued by the infectious cDNA plasmid pYS-N17D of the O-type FMDV acid-resistant mutant strain (hereinafter referred to as "rN17D mutant strain virus") and parent The one-step growth curve of this virus O/YS/CHA/05 strain.

Inoculate the parental virus and the rescued virus (rN17D mutant strain virus) into monolayer BHK-21 cells in the logarithmic growth phase according to the inoculum amount of 5MOI, absorb at 37°C for 1 hour, wash off the unadsorbed virus with PBS, add 2% FBSDMEM The culture was maintained, and the virus was harvested at 2h, 4h, 6h, 8h, 10h, 12h, 14h, and 16h, and the TCID ₅₀ of the virus harvested at different time points was measured, and each time point was repeated 3 times. Taking the virus growth time as the abscissa and the average TCID ₅₀ of the virus at different time points as the ordinate, draw a one-step growth curve of the virus.

The results of the one-step growth curve of the virus drawn are shown in Figure 4. The rN17D mutant virus has similar replication ability to the O/YS/CHA/05 virus, which shows that the VP1N17D mutation does not affect the in vitro replication ability of O-type FMDV.

Test Example 1: Immunogenicity analysis of the acid-resistant mutant strain rescued by the infectious cDNA plasmid pYS-N17D of O-type FMDV acid-resistant mutant strain

The key amino acid N17D that determines the acid-resistant phenotype of O-type FMDV is located at the N-terminal of VP1 protein, which is located outside the five antigenic sites of O-type FMDV. Therefore, it is speculated that the N17D mutation will not affect the antigenicity of the virus. In order to confirm this speculation, the foot-and-mouth disease mutant strain virus (hereinafter referred to as "rN17D mutant strain virus" that only carries the N17D site mutation) rescued by the infectious cDNA plasmid pYS-N17D of the O-type FMDV acid-resistant mutant strain was used in this test. ) and the whole virus inactivated antigen of FMD virus parental virus were treated for 30 minutes in the environment of pH7.4 and pH6.0, respectively, and Babl/c mice were immunized, serum was collected regularly, and the antibody titer was detected by neutralization test.

1. Test method

(1), inactivated vaccine immunization procedures

Seventy-five 6-week-old SPF BALB/c mice (purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were selected and randomly divided into 5 groups A-E, with 15 mice in each group. Each mouse in groups A and B was immunized with 20 μg of purified rN17D mutant virus or virus protein of the parental strain in 200 μl pH 7.4 PBS for 30 minutes, respectively. Each mouse in groups C and D was immunized with 20 μg of the above two viruses that had been purified and treated in 200 μl PBS (pH 6.0) for 30 minutes. Group E was immunized with 200 μl PBS (pH7.4) as a control. Four weeks after the first immunization, the same dose was used to boost the immunization, and at the 3rd, 6th, 9th, and 12th week after the first immunization, blood was collected from the tail vein of the immunized mice to determine the serum neutralization titer.

(2), micro-cell neutralization test

Use BHK-21 cells to measure the TCID ₅₀ of FMDVO/YS/CHA/05 virus according to conventional method; Ratio dilution; then, mix 200 TCID ₅₀ of virus with equal volumes of different dilutions of antibodies, and incubate in a 37°C incubator for 1 hour; then inoculate BHK-21 cells in the above antibody-virus mixture, 100 μL per well, each drop Repeatedly set 8 wells, culture in 5% CO ₂ incubator at 37°C, observe the cells every day, and make the final judgment after 72 hours. In addition, the virus-positive serum and normal cells were set as controls, and the antibody neutralization titer was calculated according to the Reed-Muench method according to the cytopathic effect (CPE), that is, the dilution concentration of the serum antibody that can protect 50% of BHK-21 cells from CPE.

2. Test results

The test results are shown in Table 1. The rN17D mutant virus and the parent virus O/YS/CHA/05 have similar immunogenicity after acting in a neutral (pH 7.4) environment, but in an acid (pH 6.0) environment After treatment for 30min, the immunogenicity of the rN17D mutant virus was significantly better than that of the parent virus O/YS/CHA/05.

Table 1 O type foot-and-mouth disease virus acid-resistant mutant strain inactivated antigen induced neutralizing antibody titer in mice

<110> Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences

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cgcaagatgataccgcctttcccggcgttaattggatgtaaccataagacgaaccttcac540

ccggaagtaaaacggcaaattcgcatagttttgcccgttttcaggagaaacgggacgtct600

gcgcacgaaacgcgccgtcgcttgaggaggacttgtacaaacacggtccattcaggtttc660

cacaaccgacacaaaccgtgcaacttggaactccgcctggtctttccaggtctagagggg720

tgacattttgtactgtgcttgactccacgctcggtccactggcgagtgctagtaacagca780

ctgttgcttcgtagcggagcatggtggccgcgggaactcctccttggtaacagggacccg840

cggggccgaaagccacgtcctcacggacccaccacatgtgtgcaacccccagcacggcaactt900

tattgtgaaaaccactttaaggtgacactgatactggtactcaaccactggtgacaggct960

aaggatgcccttcaggtaccccgaggtaacacgcgacactcgggatctgagaaggggact1020

ggggcttctttaaaagcgcctggtttaaaaagcttctacgcctgaataggtgaccggagg1080

ccggcacctttccttcgaacaactgtctttaaatgagcacaactgactgtttcatcgctt1140

tgttgtacgctttcagagagatcaaaacactgtttttatcacgaacgcgaggaaagatgg1200

agttcacacttcacaacggtgagaagaaaacattctactccagacccaacaaccacgaca1260

actgctggttgaacgccatcctccagctgtttaggtacgttgatgaacctttcttcgact1320

gggtctactgttcacacgagaacctcacactcaatgctataaaacaattggaagaaatta1380

ctggtctcgagctccacgagggtggaccacccgctctcgttatttggaacatcaaacacc1440

tgctcaacaccggaataggcaccgcttcgcgacccagcgaagtgtgcatggtagacggga1500

cggacatgtgcttggctgacttccatgctggcatcttcctgcaaggacaggaacacgctg1560

tgttcgcctgcgtcacctccaacgggtggtacgcaatcgatgacgaggacttttacccct1620

ggacgccggacccgtccgacgttctggtgttcgtcccgtacgaccaagaaccgctcaacg1680

gagaatggaaaacaaaggttcagaaacgactcagaggtgccgggcaatccagcccggcga1740

ctgggtcgcagaaccagtcaggtaacactggaagcattatcaacaattactacatgcagc1800

agtaccagaactccatggacacacaacttggtgacaacgctattagtggaggctccaacg1860

aggggtccacggaccacctccaccccacacaaccaacacccaaaacaacgactggtttt1920

caaagctagccagttctgcttttagcggtcttttcggcgctcttctcgctgacaagaaaa1980

ccgaggagaccactcttcttgaggaccgcatcctcactacccgcaacgggcacacgacct2040

cgacaacccagtcaagcgttggagtcacttacgggtacgcgacagctgaggactttgtga2100

gcggaccgaacacgtctgggcttgagaccagggttgtgcaggcagagcggttcttcaaaa2160

cccacttgttcgactgggtcaccagtgacccgttcggacggtgctacctgctggaactcc2220

caactgaccacaaaggtgtctacggtagcctaactgactcttatgcttacatgagaaacg2280

gttggggatgtagagggttactgcagtggggaatcagttcaacggggatgtctgttggtgg2340

ctatggtaccagaactttgctctattgacaagagaggggctttaccaactcacgctcttcc2400

cccaccagttcatcaacccccggacgaacatgacggcgcacatcactgtgccttttgttg2460

gcgtcaaccgctacgaccagtacaaggtacacagaccttggactctcgtggtcatggttg2520

tggccccgctgactgtcaacactgaaggtgccccacagatcaaggtttacgccaacatcg2580

cccctactaacgtgcacgtcgcgggtgagctcccttctaaggaagggatcttccccgtgg2640

catgtagcgacggttacggtggcctggtgaccactgacccaaagacggctgaccccgcct2700

acgggaaagtgttcaatccacctcgcaacatgttgccggggcggttcaccaacttccttg2760

atgtggctgaggcgtgtcctacgtttctgcattttgagggtgacgtaccgtacgtgacca2820

caaagacggactcagacagggtgctcgcccagtttgacttgtctctggcagcaaaacaca2880

tgtcaaacaccttcctggcaggtctcgcccagtattacacacagtacagcggcaccatca2940

acctgcacttcatgttcactggacccactgacgcgaaagcgcgttacatgattgcatacg3000

ccccccctggcatggagccgcccaaaacacccgaggcggccgctcactgcattcatgcgg3060

agtgggacacagggttgaactcaaaattcacattttcaatcccttacctttcggcggctg3120

actacgcgtacaccgcgtctgactccgcggagaccacaaacgtgcagggatgggtttgcc3180

tgtttcaaatcacacacgggaaggctgacggcgacgcgctggtcgttctagtgccg3240

gtaaggactttgaactgcgtttgccagttgatgctcgcacgcagaccacctctacaggtg3300

agtcggctgaccccgtaactgccaccgttgaggactacggtggtgagacacaggtccaga3360

gacgccagcacacggatgtctcgttcatactagacagatttgtgaaagtaacaccaaaag3420

accaaatcaatgtgttggacctgatgcaaacccctgcacacactttggtaggcgcgctcc3480

tccgtactgccacttactactttgcagatctagaagtggcagtgaaacacgagggggaacc3540

ttacctgggtcccgaatggggcgcccgaggcagcattggacaacaccaccaatccaacgg3600

cctaccacaaggcgccgctcacccggcttgcactgccttacacggcaccacaccgtgtct3660

tggctactgtttacaacgggaactgtaagtacggcaagagccccgtggccaacgcgagag3720

gtgacctgcaagtgttgaccccgaaggcggcaagaacgctgcctacctccttcaattacg3780

gcgccatcaaagccactcgggtgactgaactgctttaccgcatgaagagggccgaaacgt3840

actgcccccggcctcttttggctattcacccgagcgaaactagacacaaacaaaagattg3900

tggcgcctgtgaagcagcttttgaattttgatctgctcaagctggcaggagacgttgagt3960

ccaaccctggacccttcttcttcgctgacgtcaggtcaaatttttccaagctggttgaga4020

ccatcaaccaaatgcaggaggacatgtcaacaaaacacggacccgactttaaccggttgg4080

tgtctgcgtttgaggaactggccgctggagtgagggctatcaggactggtctcgacgagg4140

ccaaaccctggtacaagctcatcaagctactgagccgcctgtcatgcatggccgctgtag4200

cagcacggtcaaaggacccagtccttgtggccatcatgctggctgacaccggtctcgaga4260

tcctggacagtacctttgtcgtgaagaagatctccgactcgctctccagtctctttcacg4320

tgccggccccccgtcttcagtttcggagccccgattttgttggccgggttggtcaaggtcg4380

cctcgagtttcttccggtccacgcccgaagaccttgagagagcggagaaacagctcaaag4440

cacgtgacatcaatgacatattcgccattctcaagaacggcgagtggctggtcaagctga4500

tccttgccatccgcgactggatcaaggcatggatcgcctcagaggaaaagtttgtcacca4560

tgacagacctggtgcccggtatccttgaaaagcagcgggatctcaacgacccaagcaagt4620

acgaggaggccaaggagtggctcgacaacgcgcgccaagcgtgtttgaagagcgggaaca4680

tccacatcgcaaacctttgcaaagtggctgccccagcacccagcaggtcgaggcccgaac4740

ccgtggtcgtttgccttcgtggcaaatcaggccagggcaagagtttccttgcgaacgtgc4800

ttgcacaagcaatttcaacccacttcactggcagaaccgattcagtttggtactgcccac4860

ctgaccctgaccacttcgacggttacaaccagcagaccgttgtagtaatggatgacctgg4920

gccagaaccccgacgggaaggactttaagtacttcgcccaaatggtttcaactacggggt4980

ttatcccgcccatggcttcacttgaggacaaaggcaaacctttcaacagcaaggtcatca5040

tcgccaccaccaacctgtactcgggcttcaccccgagaactatggtgtgccctgatgcgc5100

tgaaccggaggttccactttgacatcgacgtgagcgctaaggacggatacaaaattaaca5160

acaaattggacatcatcaaagctcttgaagacaccaccaccaacccagtggcaatgtttc5220

aatacgactgtgcccttctcaacggcatggccgttgaaatgaagagaatgcaacaagaca5280

tgttcaagcctcaaccgcccctccagaacgtctaccagcttgttcaggaggtgattgacc5340

gggtcgagctccacgaaaaggtgtcgaaccacccgatcttcaagcagatctcaattcctt5400

cccaaaaggctgtgctgtactttctcattgagaagggccagcacgaagcagcaattgaat5460

tctttgaggggatggtgtgtgactccattaaggaggagctccggcccctaatccaacaga5520

cctcatttgtgaagcgcgcttttaagcgcctgaaggaaaactttgagattgtcgctctgt5580

gtttgacccttttggcgaacatagtgatcatgatccgcgggactcgcaagagacagcaga5640

tagtggacgatgtagtggacgagtacactgagaaggcaaacatcgccacggatgacaaga5700

ctcttgacgaggcggaaaagaaccctctggaggccagtggtgccaccactgttggtttca5760

gagagaaaactctcccgggacacaaggcgggtgatgacgtgagctctgagcccgccgaac5820

ccgtggaagggcaaccacaggctgaaggaccttacaccggtccactcgagcgtcaaaaac5880

ctctgaaagtgagagccaggctcccacagcaggaggggccctacgctggcccgatggaga5940

gacagaaaccgctgaaagtgaaagtgaaagccccggtcgttaaggaaggaccttacgaag6000

gaccggtgaagaaacctgtcgctctgaaagtgaaagcaaagaacttgattgtcactgaga6060

gtggtgctcccccgactgacttgcaaaagatggtcatgggcaacaccaagcctgttgagc6120

tcatcctcgacgggaagacggtggccatctgctgcgccaccggagtgtttggtaccgcct6180

accttgttcctcgccatcttttcgcagagaagtacgacaagatcatgttggacggcagag6240

ccatgacagacagtgactacagagtgtttgagtttgagattaaagtgaaagggcaggaca6300

tgctctcggacgccgcgctcatggtgctccaccgtgggaatcgcgtgcgggacatcacga6360

agcacttccgtgatgtggcaagaatgaagaaaggcacccccgtcgtcggcgtggtcaaca6420

acgctgatgttgggagactgatcttctctggtgaggcccttacctacaaggacattgtag6480

tgtgcatggacggagacaccatgcccggtctcttcgcctacaaagccgccaccaaggcgg6540

gttactgtggaggagccgttcttgcaaaggacggagccgagactttcatcgtcggcactc6600

actccgcaggcggcaacggagttggctactgctcgtgcgtttccaggtctatgctgctaa6660

aaatgaaggcacacatcgatcccgaaccacaccacgagggattgatagttgacaccagag6720

atgttgaggagcgcgtacatgtcatgcgcaaaaccaagctcgcacccaccgtggcatacg6780

gtgtattcaaccccgaatttggcctgccgccttgtccaaccaggacccgcgcctgaatg6840

aaggggttgtcctcgatgaagttatcttctctaaacacaaggaaaacacaaagatgtctg6900

aggaggacaaagcgctgttccgccgctgtgctgctgactacgcgtcccgcctgcacagcg6960

tgctgggtacggcaaacgccccactgagcatttacgaggcaattaagggtgtcgacggac7020

ttgacgccatggaaccagacaccgcgcctggcctcccctgggccctccaggggaaacgcc7080

gtggtgcgctcattgacttcgagaacggcactgtcggacccgaggttgaggctgctttga7140

agctcatggagaaaagagagtacaagtttgtatgccagacctttctgaaggacgagatcc7200

gtccgatggaaaaggtacgtgccggtaagactcgcattgtcgacgtcctgcctgttgaac7260

acattctttacaccaggatgatgattggtagattttgcgctcaaatgcactcaaacaacg7320

gaccgcaaattggttcggcggttggttgtaatcctgatgttgattggcaaagatttggca7380

cgcactttgctcagtacaaaaacgtgtgggatgtggactattcggcctttgacgccaacc7440

actgtagtgatgcaatgaacatcatgtttgaggaggtgttcaacacggatttcggtttcc7500

acccaaacgctgagtggatcttgaaaactctcgtggacactgaacacgcctatgagaaca7560

aacgcatcactgttgaaggcgggatgccgtctggttgttccgcgacaagcatcatcaaca7620

caattttgaacaacatctacgtgctctacgccttgcgcagacactatgagggagttgagc7680

tggactcttacaccatgatctcctacggagacgacatcgtggttgcaagtgatcacgatc7740

tggactttgaggccctcaagcctcacttcaaatcccttggtcaaaccatcactccagctg7800

acaaaagtgacaaaggttttgttcttggtcactccattaccgatgtcactttcctcaaaa7860

gacacttccacatggactatggaactgggttttacaaacctgtgatggcttcgaagaccc7920

tcgaggctatcctctcctttgcacgtcgtgggaccatacaggagaagttgatctccgtgg7980

caggactcgccgtccactctggacctgatgagtaccggcgtctctttgagcctttccagg8040

gcctcttcgagattccaagctacagatcactttacctgcgttgggtgaacgccgtgtgcg8100

gtgacgcataatccctcagatgtcacaactggcagaaagacgttgaggcgagcgacgccg8160

taggagtgaaaagtccgaaagggcttttcccgcttcctatttcaaaaaaaaaaaaaaaaaa8220

aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa8262

<210>2

<211>2208

<212>DNA

<213>artificial sequence

<400>2

ggtgccgggcaatccagcccggcgactgggtcgcagaaccagtcaggtaacactggaagc60

attatcaacaattactacatgcagcagtaccagaactccatggacacacaacttggtgac120

aacgctattagtggaggctccaacgaggggtccacggaccaccacctccaccacacaacc180

aacacccaaaacaacgactggttttcaaagctagccaattctgcttttagcggtcttttc240

ggcgctcttctcgctgacaagaaaaccgaggagaccactcttcttgaggaccgcatcctc300

actacccgcaacgggcacacgacctcgacaacccagtcaagcgttggagtcacttacggg360

tacgcgacagctgaggactttgtgagcggaccgaacacgtctgggcttgagaccagggtt420

gtgcaggcagagcggttcttcaaaacccacttgttcgactgggtcaccagtgacccgttc480

ggacggtgctacctgctggaactcccaactgcccacaaaggtgtctacggtagcctaact540

gactcttatgcttacatgagaaacggttgggatgtagaggttactgcagtggggaatcag600

ttcaacggaggatgtctgttggtggctatggtaccagaactttgctctattgacaagaga660

gggctttaccaactcacgctcttcccccaccagttcatcaacccccggacgaacatgacg720

gcgcacatcactgtgccttttgttggcgtcaaccgctacgaccagtacaaggtacacaga780

ccttggactctcgtggtcatggttgtggccccgctgactgtcaacactgaaggtgcccca840

cagatcaaggtttacgccaacatcgcccctactaacgtgcacgtcgcgggtgagctccct900

tctaaggaagggatcttccccgtggcatgtagcgacggttacggtggcctggtgaccact960

gacccaaagacggctgaccccgcctacgggaaagtgttcaatccacctcgcaacatgttg1020

ccggggcggttcaccaacttccttgatgtggctgaggcgtgtcctacgtttctgcatttt1080

gagggtgacgtaccgtacgtgaccacaaagacggactcagacagggtgctcgcccagttt1140

gacttgtctctggcagcaaaacacatgtcaaacaccttcctggcaggtctcgcccagtat1200

tacacacagtacagcggcaccatcaacctgcacttcatgttcactggacccactgacgcg1260

aaagcgcgttacatgattgcatacgccccccctggcatggagccgcccaaaacacccgag1320

gcggccgctcactgcattcatgcggagtgggacacagggttgaactcaaaattcacattt1380

tcaatcccttacctttcggcggctgactacgcgtacaccgcgtctgactccgcggagacc1440

acaaacgtgcagggatgggtttgcctgtttcaaatcacacacgggaaggctgacggcgac1500

gcgctggtcgttctagtgccggtaaggactttgaactgcgtttgccagttgatgct1560

cgcacgcagaccacctctacaggtgagtcggctgaccccgtaactgccaccgttgaggac1620

tacggtggtgagacacaggtccagagacgccagcacacggatgtctcgttcatactagac1680

agatttgtgaaagtaacaccaaaagaccaaatcaatgtgttggacctgatgcaaacccct1740

gcacacactttggtaggcgcgctcctccgtactgccacttactactttgcagatctagaa1800

gtggcagtgaaacacgagggggaaccttacctgggtcccgaatggggcgcccgaggcagca1860

ttggacaacacccaccaatccaacggcctaccacaaggcgccgctcacccggcttgcactg1920

ccttacacggcaccacaccgtgtcttggctactgtttacaacgggaactgtaagtacggc1980

aagagccccgtggccaacgcgagaggtgacctgcaagtgttgaccccgaaggcggcaaga2040

acgctgcctacctccttcaattacggcgccatcaaagccactcgggtgactgaactgctt2100

taccgcatgaagagggccgaaacgtactgcccccggcctcttttggctattcacccgagc2160

gaaactagacacaaacaaaagattgtggcgcctgtgaagcagcttttg2208

<210>3

<211>736

<212>PRT

<213>artificial sequence

<400>3

GlyAlaGlyGlnSerSerProAlaThrGlySerGlnAsnGlnSerGly

151015

AsnThrGlySerIleIleAsnAsnTyrTyrMetGlnGlnTyrGlnAsn

202530

SerMetAspThrGlnLeuGlyAspAsnAlaIleSerGlyGlySerAsn

354045

GluGlySerThrAspThrThrSerThrHisThrThrAsnThrGlnAsn

505560

AsnAspTrpPheSerLysLeuAlaAsnSerAlaPheSerGlyLeuPhe

65707580

GlyAlaLeuLeuAlaAspLysLysThrGluGluThrThrLeuLeuGlu

859095

AspArgIleLeuThrThrThrArgAsnGlyHisThrThrSerThrThrGln

100105110

SerSerValGlyValThrTyrGlyTyrAlaThrAlaGluAspPheVal

115120125

SerGlyProAsnThrSerGlyLeuGluThrArgValValGlnAlaGlu

130135140

ArgPhePheLysThrHisLeuPheAspTrpValThrSerAspProPhe

145150155160

GlyArgCysTyrLeuLeuGluLeuProThrAlaHisLysGlyValTyr

165170175

GlySerLeuThrAspSerTyrAlaTyrMetArgAsnGlyTrpAspVal

180185190

GluValThrAlaValGlyAsnGlnPheAsnGlyGlyCysLeuLeuVal

195200205

AlaMetValProGluLeuCysSerIleAspLysArgGlyLeuTyrGln

210215220

LeuThrLeuPheProHisGlnPheIleAsnProArgThrAsnMetThr

225230235240

AlaHisIleThrValProPheValGlyValAsnArgTyrAspGlnTyr

245250255

LysValHisArgProTrpThrLeuValValMetValValAlaProLeu

260265270

ThrValAsnThrGluGlyAlaProGlnIleLysValTyrAlaAsnIle

275280285

AlaProThrAsnValHisValAlaGlyGluLeuProSerLysGluGly

290295300

IlePheProValAlaCysSerAspGlyTyrGlyGlyLeuValThrThr

305310315320

AspProLysThrAlaAspProAlaTyrGlyLysValPheAsnProPro

325330335

ArgAsnMetLeuProGlyArgPheThrAsnPheLeuAspValAlaGlu

340345350

AlaCysProThrPheLeuHisPheGluGlyAspValProTyrValThr

355360365

ThrLysThrAspSerAspArgValLeuAlaGlnPheAspLeuSerLeu

370375380

AlaAlaLysHisMetSerAsnThrPheLeuAlaGlyLeuAlaGlnTyr

385390395400

TyrThrGlnTyrSerGlyThrIleAsnLeuHisPheMetPheThrGly

405410415

ProThrAspAlaLysAlaArgTyrMetIleAlaTyrAlaProProGly

420425430

MetGluProProLysThrProGluAlaAlaAlaHisCysIleHisAla

435440445

GluTrpAspThrGlyLeuAsnSerLysPheThrPheSerIleProTyr

450455460

LeuSerAlaAlaAspTyrAlaTyrThrAlaSerAspSerAlaGluThr

465470475480

ThrAsnValGlnGlyTrpValCysLeuPheGlnIleThrHisGlyLys

485490495

AlaAspGlyAspAlaLeuValValLeuAlaSerAlaGlyLysAspPhe

500505510

GluLeuArgLeuProValAspAlaArgThrGlnThrThrSerThrGly

515520525

GluSerAlaAspProValThrAlaThrValGluAspTyrGlyGlyGlu

530535540

ThrGlnValGlnArgArgGlnHisThrAspValSerPheIleLeuAsp

545550555560

ArgPheValLysValThrProLysAspGlnIleAsnValLeuAspLeu

565570575

MetGlnThrProAlaHisThrLeuValGlyAlaLeuLeuArgThrAla

580585590

ThrTyrTyrPheAlaAspLeuGluValAlaValLysHisGluGlyAsn

595600605

LeuThrTrpValProAsnGlyAlaProGluAlaAlaLeuAspAsnThr

610615620

ThrAsnProThrAlaTyrHisLysAlaProLeuThrArgLeuAlaLeu

625630635640

ProTyrThrAlaProHisArgValLeuAlaThrValTyrAsnGlyAsn

645650655

CysLysTyrGlyLysSerProValAlaAsnAlaArgGlyAspLeuGln

660665670

ValLeuThrProLysAlaAlaArgThrLeuProThrSerPheAsnTyr

675680685

GlyAlaIleLysAlaThrArgValThrGluLeuLeuTyrArgMetLys

690695700

ArgAlaGluThrTyrCysProArgProLeuLeuAlaIleHisProSer

705710715720

GluThrArgHisLysGlnLysIleValAlaProValLysGlnLeuLeu

725730735

<210>4

<211>639

<212>DNA

<213> Foot and Mouth Disease Virus

<400>4

accacctctacaggtgagtcggctgaccccgtaactgccaccgttgaggactacggtggt60

gagacacaggtccagagacgccagcacacggatgtctcgttcatactagacagatttgtg120

aaagtaacaccaaaagaccaaatcaatgtgttggacctgatgcaaacccctgcacacact180

ttggtaggcgcgctcctccgtactgccacttactactttgcagatctagaagtggcagtg240

aaacacgagggggaaccttacctgggtcccgaatggggcgcccgaggcagcattggacaac300

accaccaatccaacggcctaccacaaggcgccgctcacccggcttgcactgccttacacg360

gcaccacaccgtgtcttggctactgtttacaacgggaactgtaagtacggcaagagcccc420

gtggccaacgcgagaggtgacctgcaagtgttgaccccgaaggcggcaagaacgctgcct480

acctccttcaattacggcgccatcaaagccactcgggtgactgaactgctttaccgcatg540

aagagggccgaaacgtactgcccccggcctcttttggctattcacccgagcgaaactaga600

cacaaacaaaagattgtggcgcctgtgaagcagcttttg639

<210>5

<211>213

<212>PRT

<213> Foot and Mouth Disease Virus

<400>5

ThrThrSerThrGlyGluSerAlaAspProValThrAlaThrValGlu

151015

AspTyrGlyGlyGluThrGlnValGlnArgArgGlnHisThrAspVal

202530

SerPheIleLeuAspArgPheValLysValThrProLysAspGlnIle

354045

AsnValLeuAspLeuMetGlnThrProAlaHisThrLeuValGlyAla

505560

LeuLeuArgThrAlaThrTyrTyrPheAlaAspLeuGluValAlaVal

65707580

LysHisGluGlyAsnLeuThrTrpValProAsnGlyAlaProGluAla

859095

AlaLeuAspAsnThrThrAsnProThrAlaTyrHisLysAlaProLeu

100105110

ThrArgLeuAlaLeuProTyrThrAlaProHisArgValLeuAlaThr

115120125

ValTyrAsnGlyAsnCysLysTyrGlyLysSerProValAlaAsnAla

130135140

ArgGlyAspLeuGlnValLeuThrProLysAlaAlaArgThrLeuPro

145150155160

ThrSerPheAsnTyrGlyAlaIleLysAlaThrArgValThrGluLeu

165170175

LeuTyrArgMetLysArgAlaGluThrTyrCysProArgProLeuLeu

180185190

AlaIleHisProSerGluThrArgHisLysGlnLysIleValAlaPro

195200205

ValLysGlnLeuLeu

210

Claims (10)

1. The acidproof mutant infection cDNA cloned plasmids of 1.O type foot and mouth disease virus, is characterized in that: its Nucleotide full length sequence is for shown in SEQIDNo.1.
2. 2., according to the acidproof mutant infection cDNA cloned plasmids of O type foot and mouth disease virus according to claim 1, it is characterized in that: its deposit number is: CGMCCNO.6868.
3. 3. with the O type foot and mouth disease virus mutant strain that the acidproof mutant infection cDNA cloned plasmids of O type foot and mouth disease virus described in claim 1 or 2 is saved.
4. The acidproof mutant strain of 4.O type foot and mouth disease virus, is characterized in that: the aminoacid sequence of the capsid protein VP1 that it carries is for shown in SEQIDNo.5, and the nucleotides sequence of the described capsid protein VP1 that encodes is classified as shown in SEQIDNo.4.
5. The acidproof mutant strain of 5.O type foot and mouth disease virus, is characterized in that: the aminoacid sequence of its capsid protein carried is for shown in SEQIDNo.3, and the nucleotides sequence of described capsid protein of encoding is classified as shown in SEQIDNo.2.
6. 6.O type foot and mouth disease virus acidproof mutant strain capsid protein VP1, is characterized in that: its aminoacid sequence is for shown in SEQIDNo.5, and the nucleotides sequence of the described capsid protein VP1 that encodes is classified as shown in SEQIDNo.4.
7. 7. the purposes of the acidproof mutant infection cDNA cloned plasmids of O type foot and mouth disease virus described in claim 1 or 2 in preparation prevention or treatment aftosa vaccine or medicine.
8. 8. the purposes of O type foot and mouth disease virus mutant strain according to claim 3 in preparation prevention or treatment aftosa vaccine or medicine.
9. 9. the purposes of the acidproof mutant strain of O type foot and mouth disease virus according to claim 5 in preparation prevention or treatment aftosa vaccine or medicine.
10. 10. the purposes of O type foot and mouth disease virus acidproof mutant strain capsid protein VP1 described in claim 6 in preparation control aftosa vaccine or medicine.