O-type foot-and-mouth disease vaccine strain with improved proliferation titer as well as construction method and application of O-type foot-and-mouth disease vaccine strain

Abstract

The invention discloses an O-type foot-and-mouth disease vaccine strain with improved proliferation titer as well as a construction method and application of the O-type foot-and-mouth disease vaccine strain, and belongs to the technical field of biological products. The O-type foot-and-mouth disease vaccine strain is obtained by mutating the 144th valine (V) of structural protein VP1 into arginine (R) on a skeleton of a recombinant foot-and-mouth disease virus rHN / XJ. The replacement of the 144R of the FMDV rHN / XJ structural protein VP1 obviously shortens the time of complete CPE appearing of mutant FMDV rHN / XJ / 144R infected susceptible cells, improves the virus proliferation titer, is suitable for large-scale production, maintains the thermal stability and immunogenicity of parent viruses, and is suitable for being used as an O-type FMD vaccine candidate strain for effective prevention and control of cattle and sheep FMD in China.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an O-type foot-and-mouth disease vaccine strain with improved proliferation titer, a construction method and application thereof. Background Art

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease that is caused by the foot-and-mouth disease virus (FMDV) and is easily transmitted to major livestock such as pigs, cattle and sheep. The disease spreads quickly, has a very high incidence rate and is highly contagious. my country has classified it as a Class I animal infectious disease. The prevalence and outbreak of the disease have caused huge losses to the livestock breeding industry in the affected areas. Therefore, countries around the world attach great importance to the prevention and control of the disease.

[0003] FMDV inactivated vaccine is the vaccine with the best immune effect so far, and plays a very important role in the prevention, control and eradication of FMD. The primary factor in the screening of FMDV vaccine strains is the adaptability and proliferation performance of the virus on susceptible cells, which determines the production cost of FMD vaccines. In particular, strains with excellent proliferation performance have greater use value in production. Previous studies have shown that the genetically engineered virus rHN / KS has good cross-reactivity and excellent immunogenicity to the three currently popular O-type lineages (Mya-98 lineage, PanAsia lineage and Ind-2001 lineage) of FMDV, but has poor proliferation performance on susceptible cells and a long virus collection time, making it unsuitable as a vaccine candidate strain for cattle and sheep. The use of reverse genetic manipulation technology can improve vaccine strains, which is of great significance for improving the overall proliferation performance and antigen matching of vaccine strains.

[0004] In view of this, the present invention utilizes the reverse genetic manipulation technology of FMDV to mutate the valine (V) at position 144 of the structural protein VP1 to arginine (R) on the backbone of the recombinant FMDV rHN / XJ, thereby constructing a mutant FMDV, and studying the effect of the replacement of 144R on the proliferation performance of the mutant FMDV on susceptible cells. Summary of the invention

[0005] The purpose of the present invention is to provide an O-type FMD vaccine candidate strain with significantly improved proliferation titer and a construction method and application thereof by mutating amino acids on the FMDV structural protein VP1.

[0006] The O-type foot-and-mouth disease vaccine strain with improved proliferation titer of the present invention is obtained by mutating the 144th valine (V) of the recombinant foot-and-mouth disease virus rHN / XJ structural protein VP1 to arginine (R). The amino acid sequence of the recombinant foot-and-mouth disease virus rHN / XJ structural protein VP1 is shown in SEQ ID NO.1. For the recombinant foot-and-mouth disease virus rHN / XJ, refer to the invention patent CN2022107932887.

[0007] The method for constructing the O-type foot-and-mouth disease vaccine strain with improved proliferation titer of the present invention: (1) Using the full-length plasmid pQSA of the chimeric O / XJ / CHA / 2017 strain L+P1 gene as a template and KS144R(+) and KS144R(-) as primers, PCR amplification was performed to obtain the amplified product; The nucleotide sequence of KS144R(+) is shown in SEQ ID NO.3; the nucleotide sequence of KS144R(-) is shown in SEQ ID NO.4.

[0008] Preparation method of full-length plasmid pQSA containing L+P1 gene of chimeric O / XJ / CHA / 2017 strain: Using half-length plasmid pSK-Z123 of foot-and-mouth disease vaccine strain O / HN / CHA / 93 as template, artificially synthesize recombinant plasmid pSK-Z123XJLP1 containing L+P1 gene of foot-and-mouth disease epidemic strain O / XJ / CHA / 2017. Bgl After digestion with restriction endonucleases, the target fragment of about 5400 bp was recovered and inserted into the Bgl The full-length plasmid pQSA of the chimeric O / XJ / CHA / 2017 strain L+P1 gene was obtained from the pOFS plasmid digested with restriction endonuclease II; the nucleotide sequence of the L+P1 gene of the O / XJ / CHA / 2017 strain is as shown in SEQ ID NO.2.

[0009] The PCR amplification reaction system was as follows: 2×Max Buffer 25 μL, 10 mM dNTP Mix 1 μL, template PQSA 2 μL, 10 μM KS144R (+) 2 μL, 10 μM KS144R (-) 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, dH 2 O was added to the reaction system 50 μL.

[0010] The PCR amplification reaction conditions were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 60-72°C for 15 s, and extension at 72°C for 30-60 s, for 30 cycles; and extension at 72°C for 5 min.

[0011] (2) The amplified product was digested with DpnI enzyme and then purified and recovered by agarose gel. The DpnI digested product was then catalyzed by Exnase II enzyme to realize in vitro circularization of the linear plasmid. The product after the catalytic reaction was transformed into DH5α competent cells, single clones were selected, and plasmids were extracted for sequencing verification to obtain the mutant full-length plasmid PQSA / 144R. DpnI enzyme digestion reaction system: add 1μL DpnI enzyme to 50μL amplification product, and the digestion reaction is carried out at a constant temperature of 37℃ for 1~2 h; the in vitro cyclization reaction conditions are 37℃ for 30 min.

[0012] Exnase II enzyme-catalyzed reaction system: Dpn I digestion product 50-400 ng, 5 × CE II Buffer 4 μL, Exnase II 2 μL, ddH2O added to the reaction system 20 μL.

[0013] (3) The pQSA / 144R plasmid was used Not After I-lineage, BSR / T7 cells were transfected and the recombinant O-type foot-and-mouth disease vaccine strain rHN / XJ / 144R was rescued.

[0014] The present invention mutates the 144th valine (V) of the structural protein VP1 of the parent FMDVrHN / XJ to arginine (R), which significantly shortens the time for the infected cells to show complete CPE from the original 48 hours to 12 hours, and the replacement of 144R significantly increases the proliferation titer of the mutant FMDV on susceptible cells (more than 10 times), making it suitable for industrial production; in addition, studies on heat treatment experiments and animal experiments also show that the mutant virus rHN / XJ / 144R containing 144R has good thermal stability and immunogenicity like the parent virus rHN / XJ.

[0015] In summary, the replacement of 144R in the FMDV rHN / XJ structural protein VP1 significantly shortened the time for complete CPE to occur in susceptible cells infected with the mutant FMDVrHN / XJ / 144R, increased the virus proliferation titer, and was suitable for large-scale production. The mutant FMDV maintained the thermal stability and immunogenicity of the parental virus. The mutant virus is suitable as a candidate strain for type O FMD vaccine for the effective prevention and control of FMD in cattle and sheep in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 BSR / T7 cells with typical CPE 60h after transfection with mutant full-length plasmid; Left: normal BSR / T7 cells; Right: BSR / T7 cells 60h after transfection with pQSA / 144R; Figure 2VP1 gene fragment of transfection supernatant RT-PCR; M: 2000bp DNA marker; 1: VP1 gene fragment of PQSA transfection supernatant RT-PCR; 2: VP1 gene fragment of PQSA / 144R transfection supernatant RT-PCR; Figure 3 FMDV indirect immunofluorescence results; Figure 4 TCID of different generations of mutant FMDV 50 ; Figure 5 Thermal inactivation test for FMDV; Figure 6 Fluorescence detection of thermal dissociation curves of different FMDV particles; Figure 7 The average LPBE antibodies produced by pigs immunized with FMDV vaccine at different times. DETAILED DESCRIPTION

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Example 1 Construction of O-type foot-and-mouth disease vaccine strain 1. Construction of FMDV mutant full-length clone Using the half-length plasmid pSK-Z123 of the FMD vaccine strain O / HN / CHA / 93 as the backbone, and based on the L and P1 nucleotide sequences (SEQ ID NO.2) of the O / XJ / 2017 / CHA virus strain published in Genebank, a recombinant half-length plasmid pSK-Z123XJLP1 containing the L+P1 gene of the virus (O / XJ / CHA / 2017) was designed and synthesized (synthesized by Jin Weizhi Biotechnology Co., Ltd.). The plasmid was digested with SpeⅠ and BglⅡ restriction endonucleases to recover the target fragment of about 5400 bp, and it was inserted into the pOFS plasmid digested with the same enzymes to obtain the full-length plasmid pQSA containing the chimeric O / XJ / CHA / 2017 virus L+P1 gene. Spe Ⅰ and Bgl Ⅱ The digestion system is as follows: 10× Buffer H 10 μL, Bgl Ⅱ4 μL, Spe Ⅰ4 μL, recombinant plasmid 4 μg, ddH 2O was added to 100 μL. The enzyme digestion system was incubated at 37 °C for 1~2 h. pQSA was digested with Pst I, and the target bands of 838 bp, 4250 bp and 6050 bp were cut out, which were consistent with the expected size. The recombinant plasmids with correct enzyme digestion were sent to Jinweizhi Biotechnology Co., Ltd. for sequence determination, and the results showed that the constructed recombinant plasmid contained the expected replacement. The nucleotide sequence of the L+P1 gene of pQSA is shown in SEQ ID NO.2. The amino acid sequence of L+P1 of the O / XJ / CHA / 2017 strain is shown in SEQ ID NO.10.

[0019] The constructed FMDV chimeric full-length plasmid PQSA was used as a template to design point mutation primers KS144R(+) and KS144R(-), and the primer sequences are shown in Table 1. Then, using PQSA as a template and KS144R(+) / KS144R(-) as primers, the full-length plasmid was amplified using the Mut Express II Fast Mutagenesis Kit V2 kit, the amplification system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0020] After amplification, digestion was performed with DpnI enzyme. The reaction system was as follows: 1 μL DpnI enzyme was added to 50 μL amplification product, and the reaction was continued at 37°C for 1-2 h. The product was purified and recovered using agarose gel, and its concentration was determined. Exnase II was then used to catalyze the in vitro circularization of the linear plasmid. The reaction conditions were 37°C for 30 min, and then immediately placed on ice for cooling. The catalytic system is shown in Table 4.

[0021] The product after the catalytic reaction was completed was transformed into DH5α competent cells, and a single clone was picked and the plasmid was extracted for sequence determination. The full-length plasmid containing the expected mutation was named pQSA / 144R.

[0022] 2. Rescue of the Marked Virus The full-length recombinant plasmid pQSA and the correctly sequenced pQSA / 144R plasmid were used Not After linearization, the fragments were purified and recovered using a fragment recovery kit as a transfection template. The BSR / T7 cells that were grown in conventional culture were divided into 6 control culture plates, and the monolayer cells that grew to 80% to 90% of confluence within 24 hours were selected and lipofectamine TM 2000-mediated transfection (refer to the operating instructions for specific operations). After transfection, place at 37℃ 5% CO 2Continue culturing in the incubator and observe the cells for cytopathogenic effect (CPE) every day. Collect the transfected cell supernatant after 3-5 days of culture and set up a blank control.

[0023] The results showed that typical CPE appeared 50 hours after BSR / T7 cells were transfected with pQSA and PQSA / 144R plasmids, i.e., the fibrous cells became larger and rounder ( Figure 1 ), the control cells grew normally. The transfected cells were harvested 72 hours after transfection, and the cells were frozen and thawed three times before continuous passage on BHK-21. The viruses of each generation were stored at -70 for future use. The rescued recombinant viruses were named rHN / XJ and rHN / XJ / 144R.

[0024] The VP1 amino acid sequence of rHN / XJ is shown in SEQ ID NO.1, the VP1 nucleotide sequence of rHN / XJ is shown in SEQ ID NO.7, the nucleotide sequence of rHN / XJ / 144R is shown in SEQ ID NO.8, and the amino acid sequence of rHN / XJ / 144R is shown in SEQ ID NO.9.

[0025] 3. Identification of mutant FMDV 3.1 RT-PCR The transfection supernatant was taken and the total cytotoxic RNA was extracted using RNAasy Mini Kit. The VP1 gene of the transfection supernatant was amplified by RT-PCR using the primer pair OZ1490(+) / OZ3980(-) in the table. After purification and recovery, the gene was sent to Shanghai Sunny Co., Ltd. for sequencing to verify the correctness of the recombinant virus.

[0026] The results showed that the VP1 gene fragment was successfully amplified in the transfection supernatant (see Figure 2 ), sequencing results also showed that the rHN / XJ / 144R recombinant virus contained the expected amino acid substitution, indicating that the replacement of FMDV VP1 structural protein 144R did not affect the rescue of infectious FMDV.

[0027] 3.2 Indirect Immunofluorescence When the BHK-21 monolayer cells grew to 70% to 80% confluence, rHN / XJ and rHN / XJ / 144R recombinant viruses were inoculated respectively. The expression of 3B protein was detected by indirect immunofluorescence 6 to 8 hours after virus inoculation. The specific steps were as follows: (1) The culture medium of the infected cells was discarded, and the cells were rinsed with PBS (pH 7.6) three times, followed by addition of 4% ice-cold paraformaldehyde and fixed at room temperature for 30 minutes; (2) After rinsing with PBS three times, 5% BSA was added to block at room temperature for 30 minutes; (3) After rinsing with PBS three times, the monoclonal antibody 3B4B1 against FMDV nonstructural protein 3A (1:500 dilution) was added, and the cells were incubated at 37°C for 1 hour; (4) After rinsing with PBS five times, FITC-labeled IgG secondary antibody (1:100 dilution) was added, and the cells were incubated at 37°C for 1 hour; (5) After rinsing with PBS five times, the cells were photographed under a confocal fluorescence microscope, and normal cells were set as controls.

[0028] The results showed that BHK-21 cells inoculated with rHN / XJ and rHN / XJ / 144R could react with 3B monoclonal antibody and produce specific green fluorescence, while no visible fluorescence could be observed in the control cells when they reacted with 3B monoclonal antibody (see Figure 3 ), indicating that the present invention successfully rescued the mutated FMDV.

[0029] Example 2. Growth characteristics of mutant FMDV 1. Analysis of genetic stability of mutant FMDV The pQSA / 144R transfection supernatant was inoculated into confluent BHK-21 cells at a 10% inoculum. The cells were harvested when 95% of the inoculated cells showed typical CPE. After three freeze-thaw cycles, continuous subculture was performed. The time when 95-100% of the inoculated cells with the mutant virus rHN / XJ / 144R and the parental virus rHN / XJ showed complete CPE after the 5th generation was observed. At the same time, RT-PCR and sequence determination were performed on the 3rd and 6th generation viruses to analyze the stability of the mutant amino acids in the rescued viruses.

[0030] The results of continuous passage indicated that after the rescued mutant virus rHN / XJ / 144R was continuously passed to the fifth generation, the time for 100% of the cells to show typical CPE tended to be stable, about 12 hours, while the time for 95% of the cells to show CPE in the parental virus was about 48 hours, indicating that the replacement of the 144R structural protein VP1 of the rHN / XJ virus made the mutant FMDV adapt well to the cells, and the time for 100% of the cells to show complete CPE was significantly shortened from the original 48 hours to about 12 hours; the sequencing results showed that the mutant FMDV maintained the stability of the mutant amino acids after continuous passage to the sixth generation. The time for more than 95-100% of the cells to show complete CPE after the fifth generation of the virus is shown in Table 5.

[0031] 2. Determination of the tissue culture infectious dose (TCID) of mutant FMDV 50 ) The P5-P8 viruses of the parental virus rHN / XJ and the mutant virus rHN / XJ / 144R were diluted in 10 series and the half tissue infectious dose (TCID 50 ) method to determine the TCID of viruses of different generations 50 The experiment was repeated 3 times, with the virus TCID 50 Calculated using the Reed-Muench method.

[0032] The experimental results showed that compared with the parent virus rHN / XJ, the TCID 50 The increase was more than 10 times, indicating that the replacement of rHN / XJ virus VP1 structural protein 144R significantly improved the proliferation ability of mutant FMDV on susceptible cells.

[0033] 3. Heat-induced inactivation experiment of mutant FMDV: 3.1 Plaque assay to detect thermal stability of FMDV FMDV is sensitive to heat, especially type O FMDV. When the temperature rises, the virus particles are easy to dissociate into 12S subunits, which leads to a decrease in the content of effective vaccine antigen (146s) and affects the immune protection of the vaccine. Previous experiments have shown that rHN / XJ has good thermal stability. In order to verify whether the replacement of the 144th amino acid of the VP1 viral structural protein of the rHN / XJ virus affects the thermal stability of the mutant virus, 5x10 5PFU / mL of rHN / XJ (p6 generation), rHN / XJ / 144R (p6 generation), and heat-sensitive rHN genetically engineered virus (rHN is a recombinant virus rescued from the full-length infectious clone of FMDVO / HN / CHA / 93, disclosed in the article Evaluation of agenetically modified foot-and-mouth disease virus vaccine candidate generated by reverse genetics, BMC Veterinary Research 2012, 8:57) and heat-stable A / WH / CHA / 09 (disclosed in the article Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development, PLOS ONE 2013, Volume 8, e55228) viruses were used as controls (type A FMDV has better thermal stability than type O FMDV, and both VP2 structural proteins 93 and 98 of the strain have thermally stable amino acids) and were incubated in a 49°C water bath for 15 min, 30 min, 45 min, and 60 min, respectively. The samples were then immediately cooled on ice, and the conventional plaque assay was used to determine the toxicity (PFU / mL) of the remaining viruses after heat treatment for different times for each virus, and the percentages of remaining infectious particles of different viruses were calculated. The experiment was repeated twice.

[0034] The experimental results showed that after treatment at 49°C for 1 hour, the remaining infectious virus particles of rHN / XJ, rHN / XJ / 144R and A / WH / CHA / 09 were about 70%, while the remaining infectious virus particles of OZK / 93-08 virus were about 35% after treatment at 49°C for 15 minutes, and the remaining infectious virus particles did not exceed 10% after treatment at 49°C for 1 hour (see Figure 5 ), indicating that rHN / XJ, rHN / XJ / 144R and A / WH / CHA / 09 viruses have good thermal stability, and the replacement of VP1144R did not affect the thermal stability of the mutant FMDV.

[0035] 3.2 Thermal dissociation test of thermal stability of FMDV The above four viruses were inoculated into 100% confluent monolayer adherent BHK-21 cells (175mL cell bottle, 27ml venom solution + 3ml virus solution), and continued to be cultured in a 37°C incubator. When more than 95% of the cells showed typical CPE, the viruses were harvested separately, and about 500mL of each virus was harvested. After the collected virus solution was repeatedly frozen and thawed 3 times, it was centrifuged at 4°C 6000rpm / min for 1h to remove cell debris. The collected virus supernatant was inactivated with 1-1.2% BEI at 30°C for 28h, and the inactivated virus antigen was blindly propagated on the cells for 4 generations for safety inspection of the inactivated antigen. After passing the inspection, the virus particles were purified by sucrose density gradient centrifugation, and the 146S content of the virus antigen was detected by liquid chromatography. The purified virus was subjected to a thermal dissociation test. The reaction system was: 5 μL 100xSYBR Green Ⅱ, 500 ng~1000 ng inactivated virus particles, supplemented to 50 μL with PBS solution; reaction conditions: the initial temperature was set at 25°C, gradually increased to 99°C, each increase interval was 0.5°C, and each temperature was maintained for 10 s; the temperature at which RNA release and capsid protein were completely dissociated was monitored by fluorescence signal, and the dissociation image was drawn.

[0036] The results of the thermal dissociation experiment showed that the dissociation temperature of A / WH / CHA / 09 virus was 59.3°C, the dissociation temperature of rHN / XJ and rHN / XJ / 144R was 58.3°C, and the dissociation temperature of rHN was 53.5°C (see Figure 6 ), indicating that rHN / XJ, rHN / XJ / 144R and A / WH / CHA / 09 had higher thermal dissociation temperatures and better thermal stability, and the mutation of VP1 structural protein 144R did not affect the thermal stability of the mutant FMDV.

[0037] Example 3, preparation of FMDV inactivated vaccine Dilute the purified viral antigen to 30 μg / mL, preheat the ISA201 adjuvant in a 37°C constant temperature water bath, slowly add an appropriate amount of adjuvant to the viral antigen at a volume ratio of 1:1, and shake slowly until the antigen and adjuvant are not separated. Store the prepared vaccine product (146S antigen concentration is 15 μg / mL) at 4-8°C for later use.

[0038] Example 4: Animal Experiment Ten healthy susceptible pigs weighing about 30 kg (O-type foot-and-mouth disease liquid phase blocking ELISA antibody titer <1:6, 3ABC antibody negative) were selected and divided into two groups, 5 in each group. One group was immunized with the parental virus rHN / XJ vaccine, and the other group was immunized with the rHN / XJ / 144R virus vaccine, injected intramuscularly behind the ear root, with a dose of 2 mL / head. Blood was collected from all pigs 28 days after immunization, and serum was collected. The antibody titer of the immune serum was detected using the O-type foot-and-mouth disease virus liquid phase blocking ELISA (LPB-ELISA) kit (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences). For specific steps, refer to the kit instructions. After the experiment, the stop solution was added to stop the reaction, the OD450nm value was read on the microplate reader, the titer of each serum antibody was calculated, and the increase and decrease of the antibodies of the immunized animals were plotted. Among them, the immunized animals with an antibody titer greater than 1:128 were judged to be protected.

[0039] The results showed that the average LPBE antibody titers of pigs immunized with rHN / XJ / 144R and rHN / XJ virus vaccines were basically similar after 14, 21 and 28 days, and both vaccines could induce pigs to produce protective LPBE antibodies (>1:128) 28 days after immunization (see Figure 7 ), indicating that the replacement of structural protein VP1144R did not affect the immunogenicity of the mutant FMDV.

[0040] The research results of the present invention show that: mutating the 144th valine (V) of the structural protein VP1 of the parent FMDVrHN / XJ to arginine (R) significantly shortens the time for the infected cells to show typical CPE, which is significantly shortened from the original 48 hours to 12 hours, and the replacement of 144R significantly improves the proliferation titer of the mutant FMDV on susceptible cells (more than 10 times), and is suitable for industrial production; in addition, the research of heat treatment experiments and animal experiments also shows that the mutant virus rHN / XJ / 144R containing 144R has good thermal stability and immunogenicity like the parent virus rHN / XJ.

[0041] In summary: the replacement of FMDV rHN / XJ structural protein VP1144R significantly shortened the time for complete CPE to appear in susceptible cells infected with the mutant FMDVrHN / XJ / 144R, and increased the proliferation titer of the mutant virus on susceptible cells (more than 10 times), making it suitable for large-scale production. The mutant FMDV maintained the thermal stability and immunogenicity of the parental virus. Therefore, it is believed that the mutant virus is suitable as a candidate strain for type O FMD vaccine for the effective prevention and control of FMD in cattle and sheep in my country.

[0042] Although the present invention only achieves the improvement of the replication performance of recombinant FMDV by mutating the V at position 144 of the FMDV structural protein to R, it can be predicted that mutating the V at this position to other 18 natural amino acids may also improve the replication performance of FMDV. Therefore, any future research on FMDV mutation at position 144 to improve the viral replication performance will constitute infringement.

Claims

1. An O-type foot-and-mouth disease vaccine strain with improved proliferation titer, characterized in that: The O-type foot-and-mouth disease vaccine strain is obtained by mutating the 144th valine (V) of the recombinant foot-and-mouth disease virus rHN / XJ structural protein VP1 to arginine (R).

2. The O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 1, characterized in that: The amino acid sequence of the recombinant foot-and-mouth disease virus rHN / XJ structural protein VP1 is shown in SEQ ID NO.

1.

3. A method for constructing an O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 1, comprising the following steps: (1) Using the full-length plasmid pQSA of the chimeric O / XJ / CHA / 2017 strain L+P1 gene as a template and KS144R(+) and KS144R(-) as primers, PCR amplification was performed to obtain the amplified product; The nucleotide sequence of KS144R(+) is shown in SEQ ID NO.3; the nucleotide sequence of KS144R(-) is shown in SEQ ID NO.4; (2) The amplified product was digested with DpnI enzyme and then purified and recovered by agarose gel. The DpnI digested product was then catalyzed by Exnase II enzyme to realize in vitro circularization of the linear plasmid. The product after the catalytic reaction was transformed into DH5α competent cells, single clones were selected, and plasmids were extracted for sequencing verification to obtain the mutant full-length plasmid PQSA / 144R. (3) The pQSA / 144R plasmid was used Not After I-lineage, BSR / T7 cells were transfected and the recombinant O-type foot-and-mouth disease vaccine strain rHN / XJ / 144R was rescued.

4. The method for constructing an O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 3, characterized in that: In step (1), the preparation method of the full-length plasmid pQSA containing the L+P1 gene of the chimeric O / XJ / CHA / 2017 strain is as follows: using the half-length plasmid pSK-Z123 of the foot-and-mouth disease vaccine strain O / HN / CHA / 93 as a template, artificially synthesizing the recombinant plasmid pSK-Z123XJLP1 containing the L+P1 gene of the foot-and-mouth disease epidemic strain O / XJ / CHA / 2017, the plasmid is expressed by SpeⅠ and Bgl After digestion with restriction endonucleases, the target fragment of about 5400 bp was recovered and inserted into the Bgl The full-length plasmid pQSA of the chimeric O / XJ / CHA / 2017 strain L+P1 gene was obtained from the pOFS plasmid digested with restriction endonuclease II; the nucleotide sequence of the L+P1 gene of the O / XJ / CHA / 2017 strain is as shown in SEQ ID NO.

2.

5. The method for constructing an O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 3, characterized in that: In step (1), the PCR amplification reaction system is: 2×Max Buffer 25μL, 10 mM dNTP Mix 1μL, template PQSA 2μL, 10 μM KS144R(+) 2 μL, 10 μM KS144R(-) 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, dH2O is added to the reaction system 50μL.

6. The method for constructing an O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 3, characterized in that: In step (1), the PCR amplification reaction conditions are: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 60-72°C for 15 s, and extension at 72°C for 30-60 s, for 30 cycles; and extension at 72°C for 5 min.

7. The method for constructing an O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 3, characterized in that: In step (2), the DpnI enzyme digestion reaction system is as follows: 1 μL DpnI enzyme is added to 50 μL of amplification product, and the digestion reaction is carried out at a constant temperature of 37°C for 1 to 2 h.

8. The method for constructing an O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 3, characterized in that: In step (2), the in vitro cyclization reaction conditions are 37° C. for 30 min.

9. The method for constructing an O-type foot-and-mouth disease vaccine strain with improved proliferation titer according to claim 3, characterized in that: In step (2), the Exnase II enzyme-catalyzed reaction system: Dpn I digestion product 50-400 ng, 5 × CE II Buffer 4 μL, Exnase II 2 μL, ddH2O was added to the reaction system 20 μL.

10. Use of the O-type foot-and-mouth disease vaccine strain according to claim 1 or the O-type foot-and-mouth disease vaccine strain constructed by the method according to claim 3 in the preparation of an O-type foot-and-mouth disease vaccine.

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