CN111944770B - Attenuated mutant strain of foot-and-mouth disease virus and preparation method and application thereof - Google Patents

Abstract

The invention provides a foot-and-mouth disease virus attenuated mutant strain and a preparation method and application thereof, belonging to the technical field of biotechnology and biological products. The attenuated foot-and-mouth disease virus mutant strain is obtained by mutating Lb AUG in a parent foot-and-mouth disease virus leader protein gene into ATC or AAA, wherein the replication capacity of the mutant virus in BHK-21 cells is similar to that of a parent strain, but the replication level on PK-15 cells is reduced, the pathogenicity to suckling mice is obviously weakened, and the mutation type can be stably inherited. The invention can obviously weaken O-type foot-and-mouth disease virus after mutating 2 nd initiation codon AUG in the leader protein gene to ATC or AAA on the basis of the OZK/93-08 vaccine strain genome structure, is a method for developing genetic engineering attenuated vaccine strains, and provides a safer platform for the production of foot-and-mouth disease O-type inactivated vaccine antigens.

Description

Attenuated mutant strain of foot-and-mouth disease virus and preparation method and application thereof

technical field

The invention belongs to the technical field of biotechnology and biological products, and in particular relates to an attenuated mutant strain of foot-and-mouth disease virus and a preparation method and application thereof.

Background technique

Foot-and-mouth disease (FMD) is an acute, febrile and highly contagious animal disease caused by foot-and-mouth disease virus (FMDV), which mainly infects cloven-hoofed animals, including cattle , pigs, sheep and goats, etc. The disease is one of the animal infectious diseases legally reported by the World Organization for Animal Health (OIE). Currently, the most effective control strategies for foot-and-mouth disease are mainly achieved through vaccination with inactivated vaccines and the slaughter of infected and exposed animals. Some countries in the European Union and South America have effectively controlled foot-and-mouth disease after decades of immunization with inactivated foot-and-mouth disease virus vaccine, and are considered to be the benchmark for inactivated vaccines to prevent and control foot-and-mouth disease. In the "National Medium- and Long-Term Animal Disease Prevention and Control Plan (2012-2020)" issued in May 2012, my country clearly listed foot-and-mouth disease as a priority disease for prevention and control, and took vaccine immunization as the core technical means of prevention and control and purification.

The production of inactivated vaccines requires expensive facilities in which virulent strains of FMD virus are propagated in large numbers and there is a risk of the virus escaping from the production facility. In order to reduce the production hidden danger of scattered virus in the vaccine manufacturing process, one method is to develop a strain of foot-and-mouth disease virus with improved biosafety. The replication capacity of this engineered strain in BHK-21 cells (the seed cell line used for foot-and-mouth disease vaccine production) should not be diminished, but in other interferon-pluripotent cells or its virulence in susceptible animals Attenuated, that is, attenuated strains. Therefore, transforming virulent strains of foot-and-mouth disease vaccines from the "seed virus" source and obtaining attenuated virus strains is crucial for improving the biosafety in the vaccine production process.

The leader (leader, L) protein, a papain-like protease, is a virulence factor encoded by foot-and-mouth disease virus and plays an important role in the pathogenesis of foot-and-mouth disease virus. Therefore, the manipulation of the leader protein gene may obtain an engineered attenuated strain of FMD virus. The translation of the leader protein starts with two different start codons (Lab AUG and LbAUG) separated by 84 nucleotides (La region), which results in the existence of two forms of the leader protein, called Lab protein and Lb protein. The selection and utilization of initiation codons are one of the core steps affecting the initiation of viral protein translation and determine the translation efficiency of viral proteins. Therefore, the two initiation AUGs in the L gene play an important role in the replication of FMD virus. Previous studies have analyzed the effect of two initial AUG mutations on virus replication, and the results show that Lab AUG to UGG mutation does not prevent FMD virus rescue, but Lb AUG to UUU mutation does not rescue live FMD virus. Another study showed that insertion of a 57-nt transposon in the La region resulted in reduced availability of Lb AUG, making the virus less virulent in cattle. In addition, the insertion of an epitope tag (Flag) into the La region did not affect the survival of the virus, but eliminated or greatly reduced the activity of the virus to initiate protein translation using Lab AUG. These available results suggest that the selective use of Lab AUG and Lb AUG is critical for FMD virus replication and pathogenicity. So far, there is no Lb AUG modified FMD virus available at home and abroad, and there is no report of Lb AUG modified FMD attenuated strain.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide an attenuated mutant of foot-and-mouth disease virus, the mutant virus has an attenuated phenotype and has application potential for improving the biosafety in vaccine production.

The invention provides an attenuated mutant strain of foot-and-mouth disease virus, wherein LbAUG in the leader protein gene of foot-and-mouth disease virus is mutated to ATC or AAA.

Preferably, the nucleotide sequence of the leader protein mutated to ATC is shown in SEQ ID No.2.

Preferably, the nucleotide sequence of the leader protein mutated to AAA is shown in SEQ ID No.4.

Preferably, the nucleotide sequence of the leader protein is shown in SEQ ID No.5.

Preferably, the foot-and-mouth disease virus includes serotypes O, A, C, SAT1, SAT2, SAT3 and Asia17 foot-and-mouth disease virus.

Preferably, the O-type FMD attenuated virus strain is based on the OZK/93-08 virus strain, and the LbAUG of the leader protein is mutated to ATC or AAA.

The preparation method of the attenuated mutant strain of foot-and-mouth disease virus of the present invention comprises the following steps:

The cDNA plasmid containing the full-length genome of the foot-and-mouth disease virus is subjected to point mutation to obtain a mutated recombinant plasmid, and the mutated recombinant plasmid is transfected into cells for virus rescue to obtain a foot-and-mouth disease virus mutant strain.

Preferably, the primers for point mutation are ATC-F/ATC-R primer pair and AAA-F/AAA-R primer pair;

The nucleotide sequence of the ATC-F is shown in SEQ ID No.8;

The nucleotide sequence of the ATC-R is shown in SEQ ID No.9;

The nucleotide sequence of the AAA-F is shown in SEQ ID No.12;

The nucleotide sequence of the AAA-R is shown in SEQ ID No.13.

Preferably, the PCR amplification procedure used for the point mutation is pre-denaturation at 94°C for 5 min, 98°C for 20 min, 68°C for 1 min/1kb, 35 cycles, and then 72°C for 10 min.

The invention provides the application of the attenuated mutant strain of the foot-and-mouth disease virus in the preparation of a foot-and-mouth disease vaccine.

In the attenuated mutant strain of foot-and-mouth disease virus provided by the present invention, LbAUG in the leader protein gene of foot-and-mouth disease virus is mutated to ATC or AAA. Rescue of foot-and-mouth disease virus mutants was performed using the correctly sequenced recombinant plasmids. The results of indirect immunofluorescence test showed that two strains of LbAUG-modified O-type foot-and-mouth disease virus (rV-O/ATC and rV-O/AAA) were successfully obtained. The two mutant viruses were serially passaged on the cells, and the sequencing results showed that the two base mutations introduced in the leader protein gene could exist stably, indicating that the mutants had high genetic stability. Simultaneous plaque phenotype and one-step growth curve assays showed that rV-O/ATC and rV-O/AAA replicated similarly to the parental strain on BHK-21 cells, but rV-O on PK-15 cells The titers of /ATC and rV-O/AAA viruses were significantly lower than those of the parental viruses; while the control LbAUG was mutated to UGG or CUG, the rescued viruses rV-O/TGG and rV-O/CTG could also stably inherit, but their The replication ability is similar to that of the parental strain. The results of the virulence assay on suckling mice showed that compared with the parental strains, the LD ₅₀ of rV-O/ATC and rV-O/AAA were significantly lower, and the virulence to suckling mice was significantly weakened, while rV-O/TGG and _rV -O/CTG had slightly lower LD50s than the parental strains. The above results demonstrate that rV-O/ATC and rV-O/AAA have attenuated phenotypes. This shows that the attenuated virus strain provided by the present invention is applied to organisms or spreads into the environment, and the proliferation of the virus strain will be greatly restricted. As a vaccine strain, the attenuated virus strain has biological safety in production. will be greatly improved. Therefore, the O-type mutant strain of the foot-and-mouth disease virus prepared by the method of the present invention can be used as the seed virus of the O-type foot-and-mouth disease vaccine with improved biosafety.

Description of drawings

Fig. 1 is a schematic diagram of the full-length genome structure of LbAUG-modified foot-and-mouth disease virus mutant strain;

Figure 2 shows the indirect immunofluorescence identification of the rescued virus, in which the scale of each graph is 10 μm;

Figure 3 is an analysis of the replication ability of the rescued virus, wherein A: the plaque phenotype of the rescued virus; B: one-step growth curve of the rescued virus.

Detailed ways

The present invention provides an attenuated mutant of foot-and-mouth disease virus, wherein LbAUG in the leader protein gene of foot-and-mouth disease virus is mutated to ATC or AAA (see FIG. 1 ).

In the present invention, the nucleotide sequence of the leader protein mutated to ATC is preferably shown in SEQ ID No.2. The nucleotide sequence of the leader protein mutated to AAA is preferably shown in SEQ ID No.4. The nucleotide sequence of the leader protein is preferably shown in SEQ ID No.5.

In the present invention, since LbAUG is a conserved codon in the leader protein gene of foot-and-mouth disease virus, the two mutation schemes provided by the present invention are applicable to all serotypes of foot-and-mouth disease virus. The foot-and-mouth disease virus includes 17 serotypes of O, A, C, SAT1, SAT2, SAT3 and Asia. In order to illustrate that both mutation schemes can be used to prepare attenuated mutants of foot-and-mouth disease virus, the examples of the present invention take foot-and-mouth disease virus type O as the parental virus to conduct a specific test, but this should not be construed as a limitation on the protection scope of the present invention. When the O-type foot-and-mouth disease virus was used for mutation, the construction of the attenuated mutant of foot-and-mouth disease virus was based on the genome of the OZK/93-08 virus strain, and the LbAUG in the leader protein gene was mutated to ATC or AAA.

In the present invention, the preparation method of the attenuated mutant strain of foot-and-mouth disease virus preferably includes the following steps: on the basis of the full-length infectious clone plasmid of the foot-and-mouth disease virus strain OZK/93-08, mutating LbAUG to obtain two different mutant recombinant plasmids, Using the screened positive recombinant plasmids for virus rescue, the mutant strain of foot-and-mouth disease virus was obtained.

In the present invention, the mutation is preferably carried out using a site-directed mutagenesis technique. Primers used for point mutations are shown in Table 1. At the same time, in order to verify whether the mutation of the LbAUG site to any codon can affect the replication ability of the virus, the present invention also provides two control mutation schemes. The same, primers used for point mutation are shown in Table 1.

Table 1 Point mutation primer information

The amplification procedure of the primers was as follows: 94°C for 5 min after pre-denaturation, 98°C for 20 min, 68°C for 1 min/1 kb, after 35 cycles, 72°C for 10 min.

In the present invention, the selected positive recombinant plasmids are sequenced. The method for rescuing the virus is preferably to linearize the correctly sequenced mutant recombinant plasmid with Not I, and under the mediation of Lipofectamine ^TM 2000 (Invitrogen), transfer it into BSR/T7 cells for foot-and-mouth disease virus rescue, and the transfected cells are After incubation at 37°C for 48 hours, the supernatant was collected after freezing to obtain the rescued virus strain. After the virus strain is obtained, verification is preferably performed. The verification result is that the BHK-21 cells infected with the rescue virus can react with the foot-and-mouth disease virus MAb 3A24 antibody to produce red fluorescence, while the normal BHK-21 cells do not produce fluorescence, indicating that the foot-and-mouth disease virus can be detected during the proliferation process of the rescue virus. The synthesis of 3A protein indicates that the rescued virus is indeed a foot-and-mouth disease virus.

In the present invention, the gene mutation often leads to various forms of back mutation at the viral mutation site and changes in the viral replication ability. Therefore, the present invention performs genetic stability detection, plaque test and growth curve measurement on the two rescued viruses respectively.

The results of the genetic stability test showed that the introduced mutant base could maintain stable inheritance in the passage of the FMD virus mutant, and no other base mutation occurred in the passage of the rescue virus.

Plaque phenotypes and one-step growth curves consistently showed that rescue viruses rV-O/ATC and rV-O/AAA replicated similarly to the parental strains on BHK-21 cells (a cell line used for foot-and-mouth disease vaccine production), but The level of replication in PK-15 cells (interferon totipotent cell line) was reduced, suggesting that the 2 rescue viruses are likely to have reduced pathogenicity in FMD virus-susceptible animals.

In the present invention, since the leader protein is the most important viral protein in determining the pathogenic mechanism of foot-and-mouth disease virus, in order to further verify the influence of LbAUG mutation on the pathogenicity of foot-and-mouth disease virus, the pathogenicity test of the rescued virus in suckling mice was carried out. The results showed that compared with the parental strains, the LD ₅₀ of rV-O/ATC and rV-O/AAA was significantly lower, and the pathogenicity to suckling mice was significantly weakened, which indicated that the use of the second start codon in the mutant leader protein gene was used. This strategy can produce attenuated foot-and-mouth disease virus. Based on this, the present invention provides an application of an attenuated mutant strain of foot-and-mouth disease virus in the production of an inactivated foot-and-mouth disease vaccine.

The method for preparing the vaccine is not particularly limited in the present invention, and a vaccine preparation method well known in the art can be used.

In the event that the virus escapes the FMD vaccine manufacturing facility, such FMD virus will proliferate at a reduced rate in cells other than BHK-21 cells. Therefore, the precise engineering of foot-and-mouth disease virus by modifying the initiation codon in the leader protein gene provides a safe platform for the production of inactivated vaccine antigens.

The preparation method and application of the attenuated mutant strain of foot-and-mouth disease virus provided by the present invention will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Material source statement

BHK-21 cells were purchased from China Veterinary Supervision Institute, catalog number BHK21F5620071213, and the cells were used for virus preparation and virus titer calculation. BHK-21 cells are cells used to multiply viruses in the production of foot-and-mouth disease vaccines. This passaged cell line is an interferon-deficient cell line with limited ability to antagonize virus replication.

PK-15 is obtained through regular commodity purchases. The PK-15 cell line was able to produce and respond to interferon, and its ability to antagonize viral replication was stronger than that of the BHK-21 cell line.

BSR/T7 cells were purchased from Germany, the cells have been published, see reference "Generation of bovinerespiratorysyncytial virus (BRSV) from cDNA: BRSVNS2 is not essential for virusreplication intissue culture, and the human RSV leaderregion acts as a functional BRSV genome promoter", Journal of Virology , 1999, 73(1): 251-259, this cell was used for the rescue of foot-and-mouth disease virus.

The construction method of the full-length infectious cDNA clone plasmid pOZKF-Z1234 of foot-and-mouth disease virus OZK/93-08 strain has been disclosed (see patent CN102614507A), and the sequence of its L gene is shown in SEQ ID No.5.

Example 1

1. Construction of mutant full-length plasmids

In order to obtain the LbAUG-mutated foot-and-mouth disease virus, based on the full-length infectious cDNA clone plasmid pOZKF-Z1234 of the foot-and-mouth disease virus OZK/93-08 strain, Lb AUG was point mutated into AUC and AAA by site-directed mutagenesis, and then the LbAUG plasmids modified with different mutations. The primers used for point mutation are shown in Table 1, and the PCR amplification system is shown in Table 2. The amplification program is: 94°C pre-denaturation for 5min, 98°C 20min, 68°C 1min/1kb, after 35 cycles, 72 ℃10min. Whether the resulting recombinant plasmid introduced the expected mutation was verified by sequence determination.

Table 1 Primers used for Lb AUG point mutation

Table 2 PCR reaction system

2. Rescue mutant virus

The correctly sequenced mutant plasmid was linearized with Not I and transferred into BSR/T7 cells under the mediation of Lipofectamine ^™ 2000 (Invitrogen) for foot-and-mouth disease virus rescue. Transfected cells were incubated at 37°C for 48 hours, and the supernatant was collected after freezing.

The construction and sequence determination of mutant full-length cDNA clone showed that a full-length cDNA clone containing the expected mutation was obtained.

The correctly sequenced mutant full-length cDNA clone plasmid and wild-type (WT) plasmid were transfected into BSR/T7 cells. After 48 hours of transfection, the cells showed obvious foot-and-mouth disease virus lesions, while the cells in the control group had regular morphology and no lesions, indicating rescue. The foot-and-mouth disease virus was identified, and the rescued viruses were named rV-O/ATC and rV-O/AAA, respectively.

Comparative Example 1

The full-length plasmid and rescue mutant virus were constructed according to the method of Example 1, with different mutation schemes. LbAUG was point mutated to UGG (SEQ ID No. 1). The primers used for point mutation are shown in Table 1. The correctly sequenced mutant full-length cDNA clone plasmid was transfected into BSR/T7 cells. After 48 hours of transfection, the cells had obvious foot-and-mouth disease virus lesions, while the cells in the control group had regular morphology and no lesions, indicating that the foot-and-mouth disease virus was rescued. The virus was named rV-O/TGG.

Comparative Example 2

The full-length plasmid and rescue mutant virus were constructed according to the method of Example 1, with different mutation schemes. LbAUG was point mutated to CUG (SEQ ID No. 3). The primers used for point mutation are shown in Table 1. The correctly sequenced mutant full-length cDNA clone plasmid was transfected into BSR/T7 cells. After 48 hours of transfection, the cells had obvious foot-and-mouth disease virus lesions, while the cells in the control group had regular morphology and no lesions, indicating that the foot-and-mouth disease virus was rescued. The virus was named rV-O/CTG.

Example 2

Indirect immunofluorescence identification of rescued viruses

BHK-21 monolayer cells grew to about 70%, and the rescued parental virus (WT) and 4 strains of foot-and-mouth disease virus mutants (rV-O/TGG, rV-O/ATC, rV-O) were inoculated with an infectious dose of 10 MOI, respectively. /CTG and rV-O/AAA), incubated at 37°C for 4 h, virus-infected cells were fixed in 4% paraformaldehyde, rinsed 3 times with PBS buffer, and permeabilized with 0.5% TritonX-100 (Sigma-Aldrich) for 15 min , add 1% BSA and place it in a 37°C incubator for 1h, add foot-and-mouth disease virus 3A monoclonal antibody 3A24 dropwise, incubate at 37°C for 1h, rinse with PBS for 3 times, add FITC-labeled goat anti-mouse IgG antibody (Sigma), 37°C Incubate for 1 h, rinse with PBS for 3 times, add DAPI (Beyotime) and incubate for 15 min, rinse with PBS for 3 times, and observe the fluorescence in virus-infected cells using a laser scanning confocal microscope (LSCM).

The identification results of the rescue virus are shown in Figure 2. The BHK-21 cells infected with the rescue virus can react with the foot-and-mouth disease virus MAb 3A24 antibody to produce red fluorescence, while the normal BHK-21 cells have no fluorescence, indicating that the rescue virus is in the process of proliferation. The synthesis of FMD virus 3A protein could be detected in the rescued virus, indicating that the rescued virus was indeed FMD virus.

Example 3

Genetic stability analysis of rescued viruses

The collected supernatant was serially passaged on BHK-21 cells at a dose of 0.1 times the volume, and the 3rd, 6th, and 10th passages were taken to extract RNA, and then digested with RNase-free DNase I (Qiagen) to remove the remaining RNA. DNA followed by RT-PCR amplification to rescue the viral leader protein gene, and the resulting fragment was sequenced using primer (LCF) 5'-AGGTAACACGAGACACTC-3' (SEQ ID No. 14) to identify the genetic stability of the introduced mutated base .

The genetic stability analysis of the leader protein gene was carried out on the 3rd, 6th and 10th generation viruses of the rescued virus. The leader protein gene was amplified and sequenced using the rescued mutant virus genome as a template. The sequencing results showed that the introduced mutant base The base can maintain stable inheritance in the passage of the FMD virus mutant, and no other base mutation occurs in the rescue virus passage.

Example 4

Plaque phenotype and one-step growth curve assay of rescued virus

BHK-21 or PK-15 cells were divided into 6-well plates and cultured for 24 hours. After rinsing with PBS, 200 μL of 10× virus diluent was added to each well and incubated for 1 hour in a 37°C incubator. During this period, the cells were gently shaken every 10 minutes to avoid cell degeneration. Dry. Add 2mL of Overlay medium to each well, continue to culture for 48h, aspirate the medium, rinse the cells with PBS, add 2mL of fixative (methanol:acetone=1:1), fix at -20°C for 30min, discard the fixative, and add crystal violet staining After 30min of solution staining, the plaque morphology of the rescued virus can be observed after the staining solution is rinsed off with water. Monolayer BHK-21 or PK-15 cells were inoculated with 1MOI of rescued virus solution. After adsorption for 1h, the virus solution was aspirated and fresh medium was added. The samples were harvested at 3, 6, 9 and 20h respectively, and the TCID ₅₀ was determined to determine different time points. Virus titer in the sample.

Plaque phenotype and one-step growth curves were used to analyze the effect of LbAUG mutation on the replication ability of foot-and-mouth disease virus. As shown in Figure 3A, in BHK-21 cells, the four mutant FMD virus strains did not show large differences in plaque phenotype and plaque size compared with the parental virus. In PK-15 cells, compared with WT virus, the plaques of rV-O/TGG and rV-O/CTG were slightly reduced, but the plaques formed by rV-O/ATC and rV-O/AAA were significantly reduced . The growth curve of the mutant FMD virus (see Figure 3B) showed no significant change in the titer of the mutant FMD virus on BHK-21 cells compared to the parental virus, a result consistent with that of the plaque phenotype. In PK-15 cells, the titers of rV-O/ATC and rV-O/AAA viruses were significantly lower than those of the parental viruses, while the viral titers of rV-O/TGG and rV-O/CTG were similar to those of the parental viruses.

Example 5

The virulence test of the rescued virus in suckling mice

The 10 ^-4 to 10 ^-8 dilutions of 4 strains of FMD virus were subcutaneously inoculated into 1-day-old suckling mice (200 μl/mice, 5 animals/dilution), and the incidence of suckling mice was observed. On the 7th day, the suckling mice were counted. LD ₅₀ was calculated according to the Reed-Muench method.

The results of virulence in suckling mice showed that the LD ₅₀ of the parent virus was 10 ^-7.3 , and the LD ₅₀ of rV-O/ATC and rV-O/AAA were 10 ^-6.1 and 10 ^-6.3/ . Compared with the parental virus, the pathogenicity of rV-O/ATC and rV-O/AAA to suckling mice was significantly weakened. The LD ₅₀ of rV-O/TGG and rV-O/CTG were 10 ^-6.8 and 10 ^-6.9 respectively. Compared with the parent virus, the virulence of rV-O/TGG and rV-O/CTG in suckling mice was slightly lower. in parental strains.

Taken together, these results indicate that the rescued mutant viruses rV-O/ATC and rV-O/AAA are attenuated.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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<400> 7

gttgtgaagt gtgaattccc atttcccttg tgttcgtga 39

<210> 8

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 8

tcacgaacac aagggaaaat cgaattcaca cttcacaac 39

<210> 9

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 9

gttgtgaagt gtgaattcga ttttcccttg tgttcgtga 39

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 10

tcacgaacac aagggaaact ggaattcaca cttcacaac 39

<210> 11

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 11

gttgtgaagt gtgaattcca gtttcccttg tgttcgtga 39

<210> 12

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 12

tcacgaacac aagggaaaaa agaattcaca cttcacaac 39

<210> 13

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 13

gttgtgaagt gtgaattctt ttttcccttg tgttcgtga 39

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 14

aggtaacacg aggacactc 18

Claims (9)

1. The attenuated foot-and-mouth disease virus mutant is characterized in that an initiation codon AUG in Lb gene is mutated into ATC or AAA on the basis of O type foot-and-mouth disease virus leader protein gene.

2. The attenuated foot-and-mouth disease virus mutant strain of claim 1, wherein the nucleotide sequence of the leader protein gene mutated to ATC is shown as SEQ ID No. 2.

3. The attenuated foot-and-mouth disease virus mutant strain of claim 1, wherein the nucleotide sequence of the leader protein gene mutated to AAA is shown in SEQ ID No. 4.

4. The attenuated foot-and-mouth disease virus mutant strain of claim 1, wherein the nucleotide sequence of the leader protein gene is shown as SEQ ID No. 5.

5. The attenuated foot-and-mouth disease virus mutant of claim 1, wherein the attenuated foot-and-mouth disease virus strain O is obtained by mutating the initiation codon AUG of the leader protein gene Lb to ATC or AAA in accordance with OZK/93-08 virus strain.

6. The method for preparing the attenuated mutant strain of foot-and-mouth disease virus of any one of claims 1 to 5, comprising the steps of:

point mutation is carried out on the recombinant plasmid containing the full-length cDNA of the foot-and-mouth disease virus to obtain a mutated recombinant plasmid, and the mutated recombinant plasmid is infected with cells to be rescued to obtain a rescued virus strain.

7. The method of claim 6, wherein the primers for point mutation are ATC-F/ATC-R primer pair and AAA-F/AAA-R primer pair;

the nucleotide sequence of the ATC-F is shown as SEQ ID No. 8;

the nucleotide sequence of the ATC-R is shown as SEQ ID No. 9;

the nucleotide sequence of the AAA-F is shown as SEQ ID No. 12;

the nucleotide sequence of the AAA-R is shown as SEQ ID No. 13.

8. The method of claim 6, wherein the point mutation is performed by PCR amplification at 94 ℃ for 5min, 98 ℃ for 20min, 68 ℃ for 1min/1kb, and 35 cycles at 72 ℃ for 10 min.

9. The use of the attenuated foot-and-mouth disease virus mutant of any one of claims 1 to 5 in the preparation of a foot-and-mouth disease vaccine.

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