CN102757942A - Recombinant vaccine strain for foot-and-mouth disease type A as well as preparation method thereof and application thereof - Google Patents

Abstract

The invention relates to a type A foot-and-mouth disease recombinant vaccine strain with high titer, antigen matching and high immune protection rate constructed by gene recombination technology and its preparation method and application. The antigen nucleotide sequence of the vaccine strain is represented by SEQ ID NO: 1 It shows that the rescue system is an artificially constructed high-efficiency eukaryotic plasmid capable of expressing accurate foot-and-mouth disease virus genome RNA, thereby being able to construct and prepare foot-and-mouth disease recombinant virus, and using the above-mentioned plasmid, a vaccine strain with high titer and good antigen matching can be prepared , prepared as an inactivated vaccine, which can effectively stimulate the body to produce an immune response after immunizing pigs and cattle, and provide immune protection for pigs and cattle. In the challenge experiment of 10,000 times the bovine half infectious dose (BID ₅₀ ) of type A AISA lineage strains, The immune protection rate is 100%, and the 50% protective dose (PD ₅₀ ) is 10.81-13.59. The vaccine can be used for the prevention and control of type A foot-and-mouth disease virus in China and its surrounding countries.

Description

A Type foot-and-mouth disease recombinant vaccine strain and its preparation method and application

technical field

The present invention relates to a method for constructing a type A foot-and-mouth disease recombinant vaccine strain with high efficiency (high titer, antigen matching and high immune protection rate) with gene recombination technology, its preparation method and application, and using the vaccine strain to produce type A foot-and-mouth disease vaccine The method belongs to the field of biotechnology and biological products.

Background technique

Foot-and-mouth disease (Foot-and-mouth disease, FMD) is caused by the FMD virus (Foot-and-mouth Disease virus (FMDV) is an acute, febrile and highly contagious infectious disease caused by cloven-hoofed animals such as pigs, cattle and sheep. FMDV is a single-stranded positive-sense RNA virus belonging to the family Picornaviridae and the genus Aphthovirus. The virus is divided into 7 serotypes (A, O, C, Asian, SAT1, SAT2, and SAT3), among which type A foot-and-mouth disease virus is more prevalent and is the main epidemic strain in the world after type O foot-and-mouth disease virus. In recent years, foot-and-mouth disease epidemics have occurred frequently and are becoming globalized. This not only poses a huge threat to the world animal product economy, but also arouses people's concerns about food safety and other social public health issues. trade in livestock products suffered heavy losses.

Type A foot-and-mouth disease mostly occurs and prevails in neighboring countries of my country. In 1958, my country first discovered type A foot-and-mouth disease in the Akto area of Xinjiang. In early 1963, it was introduced from Mongolia to my country's Inner Mongolia region and caused a large-scale epidemic. The epidemic spread to more than 200 counties and cities in seven provinces of my country. In the following nearly 40 years, there was no epidemic of type A foot-and-mouth disease. However, in early 2009, my country Type A foot-and-mouth disease outbreaks broke out in Wuhan and Shanghai one after another, and soon spread to many provinces. The outbreak of type A foot-and-mouth disease has made the original complicated epidemic prevention situation in our country even more severe.

The cause of FMD antigenic variant strains is the result of antigenic diversity due to the variation of the amino acid sequence of the antigenic epitope. The antigenic epitope is an important material basis for mutagenizing the host’s immune response. How well does the antigenic epitope of the vaccine strain match the epidemic strain? Determines the level of vaccine immunization protection rate. The replication ability and virus stability of type A foot-and-mouth disease epidemic virus are low. The whole genome sequence analysis shows that the 3'UTR nucleotide and the amino acid of VP1 in the cell receptor binding site have variation, and the amino acid variation at this position may mean Changes in the replication ability of the strain and the antigenicity of the strain. Phylogenetic tree analysis showed that the outbreak of type A foot-and-mouth disease came from Southeast Asian countries, and had no genetic derivation relationship with the type A epidemic virus that had appeared in China. Vaccination is one of the main means of preventing and controlling foot-and-mouth disease. It is the most common and fast method to screen new vaccine seeds from epidemic viruses, but not every epidemic virus can be domesticated into an ideal vaccine seed virus. Immunogenicity (antigen spectrum matching, sufficient immunity induced by animal body after immunization) or production performance (toxin collection time, toxin production and virus stability) do not meet the requirements.

The ideal vaccine seed virus is hard to come by. It is urgent to innovate the vaccine seed virus screening technology and establish a long-term mechanism for vaccine seed virus renewal. The maturity of FMD reverse genetic manipulation technology has provided us with a new method to solve the screening of vaccine seed virus. It can be used to modify and modify the virus gene, artificially screen the strain with expected biological characteristics, and quickly match it with the epidemic virus. Obtain excellent vaccine strains and prepare vaccines to prevent and control epidemic strains.

Contents of the invention

The purpose of the present invention is to solve the current technical problems of lack of antigen matching and poor production performance of type A foot-and-mouth disease vaccine strains, and provide a type A foot-and-mouth disease recombinant vaccine strain and its preparation method and application, which are prepared by inactivating the vaccine strain For the vaccine, 28 days after the cattle were immunized, a 10,000-fold BID ₅₀ dose was used to challenge the virus, and the observation was continued for 12 days. The results showed that the full dose was 100% protective. The 50% protective dose (PD ₅₀ ) is between 10.81 and 13.59. This vaccine can be used for the prevention and control of type A foot-and-mouth disease virus in China and its neighboring countries.

Another object of the present invention is to provide a reverse genetic technology construction method of the above-mentioned type A foot-and-mouth disease recombinant virus vaccine strain.

Above-mentioned purpose of the present invention is achieved by following technical scheme:

A type A foot-and-mouth disease recombinant vaccine strain is characterized in that: the antigenic nucleotide sequence of the vaccine strain is shown in SEQ ID NO:1.

A reverse genetic system (plasmid) for preparing a type A foot-and-mouth disease recombinant vaccine strain is characterized in that: the system comprises: (1) a eukaryotic transcription plasmid capable of expressing the entirety of the type A foot-and-mouth disease recombinant virus Long gene cDNA sequence, the plasmid is embedded with ribozymes (hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz)), with polymerase (pol) I and pol II transcription promoters added at the 5' end of the outer side, and a terminator at the 3' end; (2) Type A Foot-and-mouth disease recombinant plasmid prA-FMDV infects sensitive cells, specifically BHK-21 cells or IBRS-2 cells. The type A foot-and-mouth disease recombinant virus is rescued by the above-mentioned reverse genetic system. The eukaryotic transcription plasmid is adapted to cells, milk Both mice and pigs were able to rescue the corresponding virus.

A kind of infectious cloning construction method of described A-type foot-and-mouth disease recombinant virus is characterized in that: take the rescue system of O-type foot-and-mouth disease virus as the skeleton, through Afl II and SgrA I restriction endonucleases, use A/WH/ The CHA/09 strain contains partial L and P1 gene sequence SEQ ID NO: 1 to replace the corresponding gene to obtain a recombinant plasmid of prA-FMDV.

The A/WH/CHA/09 strain used was isolated in Wuhan, Hubei Province in January 2009, subcultured by BHK-21 for 10 generations, and preserved in the National Foot-and-Mouth Disease Reference Laboratory designated by the Veterinary Bureau of the Ministry of Agriculture. Use the RNAeasy Mini Kit to extract the total RNA of the A/WH/CHA/09 strain, use primer olig Not I to reverse transcribe the first-strand cDNA of the virus, use the synthesized first-strand cDNA as a template, and use primers AP1-F and AP1 -R amplification to obtain the gene fragments of part L and P1 of the A/WH/CHA/09 strain, the above three specific primers for the A/WH/CHA/09 strain are:

olig Not I: 5`-ttttctaga gcggccgc t ₃₈ -3'

AP1-F: 5'-ttttc cttaag ggacaggaacatgctgtgtttgcctgcgt-3'

AP1-R: 5'-tatttt caccggtg caataattttctgcttgtgtctgt c-3',

Among the above specific primers, the upstream primer AP1-F used to amplify the partial L and P1 gene sequences of the A/WH/CHA/09 strain contains the Afl II restriction enzyme site; the downstream primer AP1-R contains the underline Part of the SgrA I restriction enzyme site was amplified with the above-mentioned specific primers to obtain part of the L and P1 gene fragments of the A/WH/CHA/09 strain, the size of which was 2400 bp, and a 50 μL reaction system was constructed. Amplification conditions For: 94°C for 5min, 94°C for 30s, 57°C for 30s, 68°C for 2min30s, go to 2, 35 cycles, 72°C for 8min, the PCR amplified product was verified by 0.8% agarose gel electrophoresis, purified and recovered and sent to Sequencing to obtain the DNA of the above gene fragments;

prO/CHA/99 is a plasmid containing the full-length cDNA of the O/CHA/99 strain constructed in our laboratory earlier. Among them, the O/CHA/99 strain is an O-type foot-and-mouth disease strain. It was isolated in Hong Kong, China in 1999 and is now Preserved in the National Foot-and-Mouth Disease Reference Laboratory, the prO/CHA/99 plasmid consists of a human cytomegalovirus RNA polymerase II promoter and a modified splice sequence encoding the bovine auxin polynucleotides signal upstream of the 5' end of the viral genome. Contains the murine RNA polymerase I promoter between the two; contains the murine polymerase terminator I and polymerase terminator II sequences downstream of the 3' end of the viral genome; and is chimeric in the O-type foot-and-mouth disease virus O/CHA/ The core sequence of Hammerase and Hepatitis Ease at both ends of the 99 full-length cDNA genome, of which Hepatitis Ease has a total of 88 ribonucleic acids, and the self-cleavage modification site is at the 5' end G; the core sequence of Hammerase has a total of 58 A ribonucleic acid, the self-cleavage modification site is at the 3' terminal C, and the infectious clone containing the O-type foot-and-mouth disease virus O/CHA/99 genome is transfected into recipient cells, through the RNA polymerase II promoter and RNA The polymerase I promoter regulatory elements were respectively transcribed and packaged to produce viral RNA precursors, and then modified and cleaved by HamRz and HdvRz to produce infectious viral RNA. The plasmid prO/ CHA/99 and the partial L and P1 fragments of the A/WH/CHA/09 strain obtained above were digested with Afl II and SgrA I respectively, ligated and transformed into JM109 competent cells, and positive clones were identified by sequencing to obtain A/WH/CHA/09 strain partial leader protein L and structural protein P1 recombinant plasmid, the recombinant plasmid is named prA-FMDV, and only site 1 is different from the circulating strain.

A kind of preparation method of described type A foot-and-mouth disease recombinant virus is characterized in that: use described recombinant plasmid prA-FMDV, direct transfection foot-and-mouth disease virus sensitive cell, preferably BHK-21 cell or IBRS-2 cell, obtain and popular Type A foot-and-mouth disease recombinant virus with matching strain antigen.

A vaccine prepared from the type A foot-and-mouth disease recombinant virus is characterized in that: after immunizing pigs and cattle, it can effectively stimulate the body to produce an immune response, and provide immune protection for pigs and cattle. In the half infectious dose (BID ₅₀ ) challenge experiment, the immune protection rate can reach 100%, and the 50% protective dose (PD ₅₀ ) is 10.81-13.59.

An application of the type A foot-and-mouth disease recombinant virus in preparing a vaccine for preventing diseases caused by the type A foot-and-mouth disease virus. The vaccine can be used for the prevention and control of the type A foot-and-mouth disease virus in my country and its surrounding countries.

The present invention has following positive effect:

The prevalence of foot-and-mouth disease type A Wuhan virus in my country puts forward a new research topic for the screening and replacement of inactivated seed virus. The cytopathic time of the virus is unstable, the titer is low, and the production requirements cannot be met. The current internationally accepted principles for screening vaccine strains are as follows: first, the antigen spectrum is matched, which can effectively protect the current epidemic strains; second, the immunogenicity is strong, which can induce animals to produce a strong enough immune response; third, the production performance is good , the vaccine candidate strain has good production performance such as short lesion time, high titer and stable lesion, and can be used in industrial production. Screening new vaccine strains from popular viruses is the most common and quickest method, which directly solves the problem of antigen spectrum matching. However, for the other two issues, immunogenicity and production performance, experiments are still needed to prove it. The reality is that not every epidemic strain can be domesticated into an ideal vaccine seed virus, and often the immunogenicity or production performance does not meet the requirements. A large number of experiments have shown that the ideal vaccine seed virus is hard to come by, and it is urgent to innovate the vaccine seed virus screening (construction) technology and establish a long-term mechanism for vaccine seed virus renewal.

The maturity of FMD reverse genetic technology provides us with a new method to solve the screening of vaccine inoculum. The transformation and modification of virus genes through reverse genetic technology can artificially obtain strains with expected biological characteristics, and can be matched with popular strains in order to match with rapidly mutating popular strains. It can minimize the negative impact of domestication of popular strains. It will lay a solid foundation for the rapid preparation of high-efficiency vaccines in the future, and is of great significance to the overall improvement of the quality of FMD vaccines. The present invention uses the established high-efficiency reverse genetic system to establish strains with short lesion time and high virus titer through the transformation of related genes, and solves the production and domestication problems of screening vaccine seed viruses. Lay the foundation for the construction of high-efficiency vaccine seeding virus.

Based on the molecular epidemiology of foot-and-mouth disease, reverse genetics was used to improve and improve the various phenotypes of FMD vaccine strains.

1) The production performance of the FMD vaccine strain (such as virus titer and lesion time) is improved at the overall level of the virus, and the strain prepared by the present invention can increase the titer by more than 100 times, reducing the cost of antigen preparation. Part of the L and P1 genes of the popular strain A/WH/CHA/09 was replaced and the recombinant virus rA-FMDV was rescued, and the biological characteristics of the virus were measured. The results showed that the recombinant virus rA-FMDV had short lesion time and high titer The characteristics of recombinant virus rA-FMDV in the 5th generation, the lesion time decreased to 11 h, from the 5th generation to the 9th generation, the complete lesion time was stable at about 10 h. While the epidemic virus A/WH/CHA/09 was from the 6th generation to the 12th generation, the complete lesion time was about 25-20 hours, and the lesion time was shortened to about 10 hours after the 13th generation. The virus titer (TCID ₅₀ ) of rA-FMDV reached 6.8 in the fifth generation, and the virus titer was stable above 7.5 from the fifth generation to the ninth generation. To the ninth generation, the virus titer was stable between 2.8 and 4.5. It can be seen that the recombinant virus has significantly improved its production performance.

2) To improve the antigen matching and immune responsiveness of the vaccine strain, the structural protein of the vaccine strain constructed by the present invention is consistent with the epidemic strain, and the antigen is completely matched, which improves the pertinence of the vaccine strain and the epidemic strain;

3) The vaccine strain prepared by the present invention contains potential vaccine identification sites, laying the foundation for the later development of labeled vaccines;

4) Compared with the vaccine prepared by the epidemic strain, the vaccine strain prepared by the present invention has improved vaccine immune response and high protection, and the PD ₅₀ value is 10.81-13.59, which enhances the field prevention and control effect of the vaccine;

6), and according to the epidemic situation in neighboring countries, with the help of the technology of the present invention, a targeted vaccine strategic reserve can be established, and a vaccine strain construction method that does not require epidemic strains (immobile virus) can be realized;

7) The technology of the present invention changes the defect that the vaccine strain selection and domestication technology is greatly restricted by the natural properties of the virus, takes time and effort, and has a low success rate, and can realize more active and effective vaccine strain construction, and realize the inactivated vaccine virus of foot-and-mouth disease The innovation of the preparation process has great application value, and the improvement of the vaccine virus seed technology belongs to the international leading level.

Description of drawings

Fig. 1 is the electrophoresis diagram of partial L and P1 gene fragments of foot-and-mouth disease virus A/WH/CHA/09 strain in Example 1 of the present invention.

Fig. 2 is the foot-and-mouth disease virus rescue system prO/CHA/99 in Example 2 of the present invention.

Fig. 3 is the construction strategy of the type A foot-and-mouth disease virus recombinant plasmid prA-FMDV in Example 2 of the present invention.

Fig. 4 is a diagram showing the comparison of site 1 differences between the recombinant strain of Example 2 of the present invention and the prevailing strain.

Fig. 5 is the CPE caused by rescuing recombinant virus rA-FMDV from infection of BHK-21 cells in Example 3 of the present invention.

Fig. 6 is an indirect immunofluorescence image of BHK-21 cells inoculated with the rescue recombinant virus rA-FMDV in Example 4.1 of the present invention.

Fig. 7 is a comparison chart of the pathogenicity test of the rescued recombinant virus rA-FMDV and the epidemic strain A/WH/CHA/09 on BHK-21 cells in Example 5.2 of the present invention.

Detailed ways

The present invention combines the accumulation of virus strains in the National Foot-and-Mouth Disease Reference Laboratory in recent years and the analysis and research on its whole genome sequence, and the part of the leader protein L and structural protein P1 gene of my country's Type A foot-and-mouth disease strain A/WH/CHA/09, through The restriction endonuclease site is replaced with the corresponding nucleotide sequence in the rescue system of the established O-type foot-and-mouth disease virus O/CHA/99 strain, and a kind of A-type foot-and-mouth disease virus recombinant plasmid prA-FMDV is obtained; the specificity The nucleotide sequence is shown in SEQ ID NO:1.

A reverse genetic operating system for preparing a type A foot-and-mouth disease recombinant vaccine strain, the system comprising: (1) a eukaryotic transcription plasmid, which can express the full-length gene cDNA sequence of the recombinant type A foot-and-mouth disease virus, and the plasmid is in the whole virus The long cDNA is flanked by ribozymes (hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz)), with polymerase (pol) I and pol II transcription promoters added at the 5' end of the outer side, and a terminator at the 3' end; (2) Type A The foot-and-mouth disease recombinant plasmid prA-FMDV infects sensitive cells, preferably BHK-21 cells or IBRS-2 cells. The type A foot-and-mouth disease recombinant virus is rescued by the above-mentioned rescue system, and the plasmid can rescue the corresponding virus in adapted cells, suckling mice and pigs.

The infectious clone of type A foot-and-mouth disease recombinant virus is based on the rescue system of type O foot-and-mouth disease virus vaccine strain, through Afl II and SgrA I restriction enzymes, using A/WH/CHA/09 strain containing part of L and The P1 gene was replaced with the corresponding gene to obtain the recombinant plasmid of prA-FMDV.

A method for preparing a type A foot-and-mouth disease recombinant virus, using the recombinant plasmid to directly transfect foot-and-mouth disease virus-sensitive cells, preferably BHK-21 cells or IBRS-2 cells, to obtain a type A foot-and-mouth disease recombinant virus that matches the epidemic strain antigen , the type A foot-and-mouth disease recombinant virus prepared by the method is named as rA-FMDV.

The construction method of the above-mentioned type A foot-and-mouth disease virus recombinant plasmid prA-FMDV is to use specific primers to amplify the genome of the type A foot-and-mouth disease virus strain A/WH/CHA/09 to obtain part of the leading protein L and structural protein P1 genes. The plasmid prO/CHA/99 of type O foot-and-mouth disease virus was replaced by specific restriction sites of Afl II and SgrA I, and the recombinant plasmid of type A foot-and-mouth disease virus was constructed and named prA-FMDV. Cytopathic pathology (CPE) appeared in the second passage of transfected BHK-21 cells for 48 hours, and the rescued FMDV was detected by RT-PCR, indirect immunofluorescence, reverse indirect hemagglutination, and pathogenicity experiments in suckling mice after continuous passage. Compared with the epidemic virus, the recombinant virus rA-FMDV has the characteristics of shorter lesion time and higher virus titer after the measurement of biological characteristics.

In the application of the above-mentioned type A foot-and-mouth disease recombinant virus vaccine strain, the genetically engineered virus obtained from the infectious clone of the type A foot-and-mouth disease recombinant virus was inactivated to make a vaccine, and the measured PD ₅₀ of the vaccine was between 10.81 and 13.59. Solve the problems in actual production, and obtain the improved type A foot-and-mouth disease High-efficiency vaccine strain. Through the detection of the virus-carrying status after the challenge, the diseased cattle can detect the virus-carrying, while the immune-protected cattle did not form the virus-carrying phenomenon. Based on the above indicators, the recombinant strain is a high-efficiency vaccine strain.

The present invention will be further described below in conjunction with specific implementation examples, but the specific implementation does not limit the present invention in any way.

The experimental methods used in the following examples, unless otherwise specified, are conventional conditions, such as "Refined Molecular Biology Experiment Guide" (F.M. Osper, R.E. Kingston, J.G. Seidman, etc. editors, Ma Xuejun, Shu Yuelong Translated by the method described in Beijing: Science Press, 2004).

Example 1. Obtainment of partial L and P1 gene sequences of type A foot-and-mouth disease virus A/WH/CHA/09 strain.

The A/WH/CHA/09 virus strain used by the inventor is preserved by the National Foot-and-Mouth Disease Reference Laboratory designated by the Veterinary Bureau of the Ministry of Agriculture, and the public can obtain it through a letter of entrustment issued by the Veterinary Bureau of the Ministry of Agriculture. Use the RNAeasy Mini Kit (Qiagen Company) to extract the total RNA of the A/WH/CHA/09 strain, use the primer olig Not I to reverse transcribe the first-strand cDNA of the virus, use the synthesized first-strand cDNA as a template, and use the primer AP1 -F and AP1-R were amplified to obtain the gene sequence of A/WH/CHA/09 strain. The above three specific primers for strain A/WH/CHA/09 are olig Not I (5'-ttttctaga gcggccgc t ₃₈ -3'), AP1-F (5'-ttttc cttaag ggacaggaacatgctgtgtttgcctgcgt-3') and AP1- R (5'-tatttt caccggtg caataattttctgcttgtgtctgt c-3'). Among the above specific primers, the upstream primer AP1-F used to amplify the partial L and P1 gene sequences of the A/WH/CHA/09 strain contains Afl II restriction enzyme site; the downstream primer AP1-R contains SgrA I Restriction site (underlined part). Using the above-mentioned specific primers to amplify, the partial L and P1 gene fragments of the A/WH/CHA/09 strain were obtained, with a size of 2400bp (Figure 1), which was consistent with the expected size. The DNA polymerase used for amplification is Premix LA Taq®Version 2.0 (TaKaRa Company) with excellent performance, which is suitable for amplification of long fragments. Construct a 50 μL reaction system according to the product manual. The amplification conditions are: 94°C for 5min, 94°C for 30s, and 57°C for 30s , 68°C 2min30s, go to 2, 35 cycles, 72°C 8min, the PCR amplification product was purified and recovered after being verified by 0.8% agarose gel electrophoresis and sent for sequencing to obtain the DNA of the above gene fragment; The results of electrophoresis are shown in Figure 1.

Example 2. Construction of infectious clones of type A foot-and-mouth disease recombinant virus.

Based on the framework of the O-type foot-and-mouth disease virus O/CHA/99 strain rescue system prO/CHA/99, the O-type foot-and-mouth disease virus strain rescue system prO/CHA/99 is shown in Figure 2: at the 5' end of the virus genome The upstream contains the human cytomegalovirus RNA polymerase II promoter (Human cytomegalovirus RNA polymerase II promoter, P _II ) and the modified splicing sequence encoding the bovine auxin polynucleotide acidification signal, and the mouse RNA polymerase is contained between them I promoter (Mouse RNA polymerase I promoter, P _I ); in the downstream of the 3' end of the viral genome, it contains murine polymerase terminator I (Murine terminator I, T _I ) and polymerase terminator II (Murine terminator II, T _II ) sequence; and the core sequences of Hammerhead ribozyme (HamRz) and Hepatitis delta ribozyme (HdvRz) chimeric at both ends of the full-length cDNA genome of type O foot-and-mouth disease virus O/CHA/99, of which type E The hepatitis enzyme has 88 ribonucleic acids in total, and the self-cleavage modification site is at the 5' terminal G; the hammer hammer enzyme core sequence has a total of 58 ribonucleic acids, and the self-cleavage modification site is at the 3' terminal C. The infectious clone containing the O/CHA/99 genome of type O foot-and-mouth disease virus is transfected into recipient cells, and the viral RNA precursors are transcribed and packaged through the RNA polymerase II promoter and the RNA polymerase I promoter regulatory elements respectively, and then Modified cleavage by HamRz and HdvRz produces infectious viral RNA.

After the plasmid prO/CHA/99 of the O-type foot-and-mouth disease virus O/CHA/99 strain rescue system and the part of L and P1 fragments obtained in Example 1 were digested with Afl II and SgrA I respectively, they were ligated and transformed into JM109 competent cells , positive clones were identified by sequencing, a recombinant plasmid containing part of the leader protein L and structural protein P1 of the A/WH/CHA/09 strain was obtained, and the recombinant plasmid was named prA-FMDV (Figure 3), and only site 1 was related to the epidemic virus strains were different (Figure 4).

Example 3. Rescue of type A foot-and-mouth disease recombinant virus.

The recombinant plasmid prA-FMDV containing the whole genome cDNA of type A foot-and-mouth disease recombinant virus obtained by Example 2 was purified and prepared with QIAGEN ^® Plasmid Plus Maxi Kit produced by QIAGEN. When the BHK-21 monolayer grows to 80%-90%, it is used for transfection. Wash the cells with OPTI-MEMI medium, transfect 4 μg of the recombinant plasmid into BHK-21 cells under the mediation of liposome Lipofectamine ^TM 2000 (Invitrogen), and place them in a 37°C incubator containing 5% CO2 Culture, and set up liposome control and normal cell control at the same time. 4-6 hours after transfection, the cell supernatant was aspirated, and MEM medium (Invitrogen Company) was added, and the culture was continued to observe the occurrence of cell lesions; the virus was harvested when about 90% of the cells were lesions. After repeated freezing and thawing 3 times, BHK-21 cells were inoculated again until the virus could stably produce CPE, the cells became round and distributed in grape shape, and finally the cells disintegrated into fragments (as shown in Figure 5). The type A foot-and-mouth disease recombinant virus was named rA-FMDV. In Fig. 4, A: a picture of CPE that appeared for rescue of recombinant virus-infected BHK-21 cells; B: picture of normal control BHK-21 cells.

Example 4. Rescue identification of type A foot-and-mouth disease recombinant virus.

4.1 Detection of virus antigen by indirect immunofluorescence.

After transfection, the BHK-21 cells that were blindly passed to the second generation were frozen and thawed three times at -70°C, and then inoculated in a certain proportion into a six-well plate with BHK-21 cells on the bottom with slides (single Layer cells grow to 60% ~ 70%), placed in 37 ℃ incubator containing 5% CO2, after 24 hours, indirect immunofluorescence was performed according to the conventional method, the primary antibody was positive serum of type A FMDV rabbits, and the secondary antibody was labeled FITC Goat anti-rabbit IgG (Sigma Company), and a normal cell control was set at the same time. Green-specific fluorescence can be seen in BHK-21 cells inoculated with the second-generation cell fluid, but no visible fluorescence can be seen in the normal cell control (as shown in Figure 6, A: BHK-21 cells inoculated with the rescue recombinant virus rA-FMDV; B: Normal BHK-21 cells not inoculated with virus), indicating that there is expression of FMDV protein in BHK-21 cells infected with recombinant virus.

4.2 Reverse indirect hemagglutination identification test

Collect 1ml of the 7th-generation rescued virus that was passaged on BHK-21 cells when CPE appears to be stable, inactivate at 58°C for 40 minutes, and serially dilute the inactivated virus at 1:2 (set blank control and epidemic at the same time). Virus A/WH/CHA/09 positive control), 50 µL of each dilution was added to a 96-well plate, and then 25 µL of FMDV antibody-sensitized sheep red blood cells were added, shaken on a micro mixer for 1-2 min, Cover and observe the results after standing at room temperature for 2 h. When the FMDV antibody attached to the blood cells meets the free antigen, the antigen-antibody agglutination network is formed, and the sheep red blood cells also agglutinate, and the hemagglutination phenomenon visible to the naked eye appears, so the result can be judged according to the cell deposition.

The results showed that when the rA-FMDV diluted at 1:32 infected BHK-21 cells, erythrocyte agglutination occurred, but no erythrocyte deposition occurred; 75% of the prevalent virus diluted 1:32 appeared erythrocyte agglutination, and the negative control samples showed complete deposition of erythrocytes; The results of indirect hemagglutination test showed that: FMDV is the cause of BHK-21 cell lesions.

Example 5. The pathogenicity test of rescuing type A foot-and-mouth disease recombinant virus.

5.1 Pathogenicity test on suckling mice.

Dilute the rescue virus and the epidemic virus that were propagated to the 6th passage in BHK-21 cells respectively with PBS buffer solution 10 times, and each take ^10-5-10-9 times diluted virus solution and inoculate it subcutaneously for 2-4 days ^. Four days-old suckling mice were inoculated for each dilution, and the inoculation dose was 200 μL/mouse, and they were continuously observed for 8 days. Four suckling mice in the blank control group were inoculated with 200 μL of PBS buffer per mouse, the death of suckling mice was observed and recorded, and the LD ₅₀ was calculated by Karber method. Some of the suckling mice inoculated with the rescue virus and the epidemic virus showed symptoms such as dyspnea, hindlimb paralysis, and hoarse voice after pinching the tail with tweezers 20 hours after inoculation. After 48 hours, the suckling mice inoculated with the epidemic virus and rescued recombinant virus died one after another, while the suckling mice in the control group were normal. Finally, according to the statistical results, the LD ₅₀ of the rescue virus rA-FMDV was calculated to be 10 ^-7.5 /0.2mL, and the LD ₅₀ of the epidemic strain A/WH/CHA/09 was 10 ^-5.0 /0.2mL.

5.2 Pathogenicity test on BHK-21 cells.

Digest BHK-21 cells according to conventional methods, add MEM cell culture medium containing 10% fetal bovine serum, divide into 28 2mL cell culture flasks, and culture them in an incubator containing 5% CO2 at 37°C. Standby when the single layer grows to 90%. Use MEM to dilute the virus solution by 10 times, and add the virus solution of each dilution (10 ^-3.0 to 10 ^-8.0 ) into 2mL cell culture bottles, 4 bottles for each dilution, and inoculate at 10% at 37°C for infection. For 1 hour, wash the cells with serum-free MEM for 2 to 3 times, add serum-free MEM, put them in an incubator containing 5% CO2 at 37 °C for cultivation, observe for 3 days, and use the Read-Muench method to determine the Half infectious dose (TCID ₅₀ ) of BHK-21 cells. According to this method, the titer of the rescued virus rA-FMDV and the 1-9 generation of the parental virus was determined, and the lesion time of each generation was counted.

After the rescue virus rA-FMDV was inoculated into BHK-21 cells, the cells were subcultured to the fifth passage, and the lesion time was reduced to 11 hours, and the complete lesion time was stable at about 10 hours from the fifth to the ninth passage; In the 12th generation, the complete lesion time was about 25-20 hours, and after the 13th generation, the lesion time was shortened to about 10 hours. The TCID ₅₀ test was carried out with the poison, and the BHK-21 cells in the blank control group were cultured for 24 hours to cover the 96-well cell culture plate, which met the experimental requirements. When the experimental group was cultured for 4 hours, most of the cells at various dilutions showed pathological changes, and after 9-14 hours of culture, all the cells fell off. Calculated by the Read-Muench method, the modified genetically engineered virus had a titer of 6.8 in the 5th generation, and the virus titer was stable above 7.5 from the 5th to the 9th generation. The parental virus titer was 2.8 at the fifth generation, and the virus titer was stable between 2.8 and 4.5 from the fifth generation to the ninth generation (as shown in Figure 8).

Example 6. The preparation and security inspection of the rescue type A foot-and-mouth disease recombinant virus vaccine.

BHK-21 was cultured and passaged in DMEM containing 10% fetal bovine serum. The rA-FMDV rescued virus liquid was prepared by the conventional spinner bottle monolayer BHK-21 passage cell culture method, and the harvested virus liquid was the virus culture, which was stored at -40°C. Inactivate the virus culture with 3 mmol/l Diethyleneimine (Binary ethyleneimine, BEI) (Sigma Company) was inactivated at 26°C, and BEI was added after 24 hours, and then inactivated for another 24 hours. After inactivation, security inspection, antigen concentration and purification, the antigen harvest and ISA206 adjuvant (SEPPIC, France) were mixed at a ratio of 1:1 to prepare a vaccine. Specifically, it is prepared according to the regulations of the foot-and-mouth disease inactivated vaccine for veterinary biological products in the Pharmacopoeia of the People's Republic of China. During the security inspection, 2ml of inactivated virus was injected subcutaneously into the tongue of the cattle, and the cattle were observed continuously for 6 days. During the observation period, the cattle were in good health, and there were no abnormalities in the hooves, mouth and nose.

The cattle used in the above experiments were purchased from non-epidemic areas, and the FMD liquid-phase blocking ELISA (LPB-ELISA) produced by the National Foot-and-Mouth Disease Reference Laboratory was used to detect O-type and AsiaI-type antibodies with a ratio of <1:4. FMD nonstructural protein 3ABC-ELISA antibody test was negative.

Example 7. Comparison of the effects of vaccine immunization on animals.

7.1 Vaccine immune challenge protection test

The qualified A-type foot-and-mouth disease recombinant virus vaccine obtained in the above-mentioned embodiment 6 was immunized with 16 cows, and 2 non-immune cows were compared at the same time (required that the anti-enemy titer of the immunized cows was not less than 1:6, and the infection antibody was negative), and its immune efficacy was determined. The methods of challenge and result determination were in accordance with the "Manual of diagnostic tests and vaccines for terrestrial animals" (2009 edition, World Organization for Animal Health (OIE)). 28 days after the cattle were immunized, the challenge experiment was carried out with 10,000 times the ID ₅₀ dose of the virus, and the observation was continued for 12 days. The results showed that: using the type A foot-and-mouth disease recombinant virus as a vaccine strain, it was inactivated after being propagated on the cultured BHK-21 cells. ISA206 adjuvant (SEPPIC, France) is emulsified into a vaccine to immunize cattle, which can effectively protect against the current type A foot-and-mouth disease epidemic virus in China. The results are shown in Table 1.

surface 1A Clinical symptoms and protective status of cattle immunized with recombinant foot-and-mouth disease virus vaccine.

7.2 Cross-immune neutralization experiment.

The serum obtained by the A-type foot-and-mouth disease recombinant virus vaccine qualified in the above-mentioned embodiment 6 obtained from the animal and rA-FMDV, A/WH/CHA/09 and O/CHA/99 three-strain virus are carried out in virus cross-immunization And experiment, determine the antibody matching value r, where 1≥r≥0.3 indicates that the strain is highly compatible with the vaccine, and the vaccine can resist the attack of this strain after immunizing animals, which can be used as a potential vaccine strain; r≤0.3 indicates that the strain is compatible with the vaccine The vaccine is poorly matched, and the vaccine cannot resist the attack of the corresponding strain after the animal is immunized. The results are shown in Table 2.

surface 2 . Virus cross immune neutralization test rA-FMDV, A/WH/CHA/09 and O/CHA/99 Antibody matching value.

7.3 Vaccine immunity test (PD ₅₀ )

The cattle used in the test were purchased from non-epidemic areas, and the type A antibodies were all <1:4 in the FMD liquid-phase blocking ELISA (LPB-ELISA) produced by the National Foot-and-Mouth Disease Reference Laboratory. FMD nonstructural protein 3ABC-ELISA antibody test was negative. All animals used in this experiment were raised strictly in the ABSL-3 laboratory. The 72 experimental cattle were divided into 4 groups. The control group was immunized with the vaccine prepared by the epidemic virus A/WH/CHA/09, and the experimental group was immunized with the vaccine prepared by the recombinant rA-FMDV virus. The challenge virus was 10,000 times BID50 The epidemic virus A/WH/CHA/09. The way of attacking the poison is to inject the venom at multiple points in the skin of the tongue. Continuous observation for 30 days, according to the symptoms of foot-and-mouth disease such as vesicles and ulcers on the tongue, gums and hooves of the cattle, it was determined whether the virus liquid caused the disease of cattle, so as to determine the toxicity of the cytovenom to cattle. The results of the immune efficacy test showed that the PD50 of the recombinant strain was 10.81-13.59, while the PD50 of the vaccine prepared by the epidemic strain was 5.57 (Table 3).

surface 3 Recombinant strains and circulating strains _PD50 Experiment Comparison Table

Example 8. Virus-carrying detection in vaccine immunized animals.

Two methods of non-structural protein 3ABC antibody ELISA and viral nucleic acid real-time quantitative PCR were used for monitoring. The real-time quantitative nucleic acid was quantified using TaqMan PCR technology, and the primers SA- IR-219-246F: 5`-CACYTYAAGRTGACAYTGRTACTGGTAC-3; SA-IR-315-293R: 5`-CAGATYCCRAGTGWCICITGTTA-3` and probe SAmulti2-P-IR-292-269R: 5`-CCTCGGGGTACCTGAAGGGCATCC-3` is used for rapid, sensitive and specific detection of virus-carrying status in blood and OP solution 32 days after immune challenge. The results showed that the sick animals were able to produce 3ABC antibodies, and the viral nucleic acid could be monitored. However, immune protection animals cannot detect antibodies and nucleic acids (Table 4, 5)

Table 4 Nonstructural protein 3ABC antibody ELISA detection of antibody results in serum animal number 0 dpc 7 dpc 14 dpc 21 dpc 28 dpc 32 dpc 1251 - - + + + + 1252 - - - - - - 1256 - - + + + + 1262 - - - - - - 1266 - - + + + + 1269 - - - - - - 1274 - - - - - - 1287 - - - - - - 1255 - - - - - - 1259 - - - - - - 1264 - - - - - - 1277 - - - - - - 1278 - - - - - - 1279 - - - - - - 1282 - - - - - - 1286 - - - - - - 1265 - + + + + + 1280 - + + + + +

dpc: Indicates the number of days after immune attack

+ indicates a positive sample; - indicates a negative sample

surface 5 Real-time quantification of viral nucleic acid PCR Test results

animal number blood nasal fluid OP solution 1251 - - 7(23.6) 1252 - - - 1256 - - 7(21.5) 1262 - - - 1266 - - 7(25.5) 1269 - - - 1274 - - - 1287 - - - 1255 - - - 1259 - - - 1264 - - - 1277 - - - 1278 - - - 1279 - - - 1282 - - - 1286 - - - 1265 2(32.4) 2(30.4) 5(31.2) 7(23.5) 14(30.1) 28(31) 32(33) 1280 2(36.5) 2(33.14) 5(35.2) 7(24.1)

+ indicates a positive sample; - indicates a negative sample

The number indicates the number of days after immune challenge, and the number in brackets indicates the ct value (ct value less than 35 is a positive sample)

The above-mentioned embodiments only describe the implementation of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make other improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

The present invention relates to a kind of reverse genetic technology to prepare recombinant vaccine strain of type A foot-and-mouth disease and its method for producing vaccine. and preparation of foot-and-mouth disease recombinant virus. Vaccine strains with high titers and good antigen matching can be prepared by using the above plasmids, which can be prepared as inactivated vaccines, which can effectively stimulate the body to produce immune responses after immunizing pigs and cattle, and provide immune protection for pigs and cattle. In the 10,000 times bovine half infectious dose (BID ₅₀ ) challenge experiment of the virus strain, the immune protection rate reached 100%, and the 50% protective dose (PD ₅₀ ) was 10.81-13.59. The vaccine can be used for the prevention and control of type A foot-and-mouth disease virus in my country and its neighboring countries.

Claims (7)

1. A type A foot-and-mouth disease recombinant vaccine strain, characterized in that: the antigenic nucleotide sequence of the vaccine strain is shown in SEQ ID NO:1. 2. A reverse genetic system for preparing type A foot-and-mouth disease recombinant vaccine strain as claimed in claim 1, characterized in that: the reverse genetic system comprises: (1) eukaryotic transcription plasmid, which can express The full-length gene cDNA sequence of type A foot-and-mouth disease recombinant virus, the plasmid is embedded with ribozymes (hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz)) on both sides of the full-length cDNA of the virus, and a polymerase ( polymerase, pol) I and pol II transcriptional promoters, with terminators at the 3' end; (2) Type A foot-and-mouth disease recombinant plasmid prA-FMDV infects sensitive cells, specifically BHK-21 cells or IBRS-2 cells, the A Type FMD recombinant virus was rescued by the above-mentioned reverse genetic system, and the eukaryotic transcription plasmid could rescue the corresponding virus in adapted cells, suckling mice and pigs. 3. an infectious cloning construction method of A-type foot-and-mouth disease recombinant virus as claimed in claim 2, is characterized in that: take the rescue system of O-type foot-and-mouth disease virus as skeleton, by Afl II and SgrA I restriction endonuclease , replace the corresponding gene with the sequence SEQ ID NO: 1 containing part of the L and P1 genes in the A/WH/CHA/09 strain to obtain the recombinant plasmid of prA-FMDV. 4. The method for constructing infectious clones of A-type foot-and-mouth disease recombinant virus according to claim 3, characterized in that: the A/WH/CHA/09 strain used was isolated in Wuhan, Hubei in January 2009, and passed down through BHK-21 Cultured for 10 generations, preserved in the designated preservation unit of the Veterinary Bureau of the Ministry of Agriculture: National Foot-and-Mouth Disease Reference Laboratory, using the RNAeasy Mini Kit to extract the total RNA of the A/WH/CHA/09 strain, and using the primer olig Not I to reverse transcribe and synthesize the virus The first Strand cDNA, using the synthesized first-strand cDNA as a template, using primers AP1-F and AP1-R to amplify the gene fragments of part L and P1 of the A/WH/CHA/09 strain, the above three are for A/WH/CHA /09 strain specific primers are: olig Not I: 5`-ttttctaga gcggccgc t ₃₈ -3' AP1-F: 5'-ttttc cttaag ggacaggaacatgctgtgtttgcctgcgt-3' AP1-R: 5'-tatttt caccggtg caataattttctgcttgtgtctgt c-3', Among the above specific primers, the upstream primer AP1-F used to amplify the partial L and P1 gene sequences of the A/WH/CHA/09 strain contains the Afl II restriction enzyme site; the downstream primer AP1-R contains the underline Part of the SgrA I restriction enzyme site was amplified with the above-mentioned specific primers to obtain part of the L and P1 gene fragments of the A/WH/CHA/09 strain, the size of which was 2400 bp, and a 50 μL reaction system was constructed. Amplification conditions For: 94°C for 5min, 94°C for 30s, 57°C for 30s, 68°C for 2min30s, go to 2, 35 cycles, 72°C for 8min, the PCR amplified product was verified by 0.8% agarose gel electrophoresis, purified and recovered and sent to Sequencing to obtain the DNA of the above gene fragments; prO/CHA/99 is a plasmid containing the full-length cDNA of the O/CHA/99 strain constructed in our laboratory earlier. Among them, the O/CHA/99 strain is an O-type foot-and-mouth disease strain. It was isolated in Hong Kong, China in 1999 and is now Preserved in the National Foot-and-Mouth Disease Reference Laboratory, the prO/CHA/99 plasmid consists of a human cytomegalovirus RNA polymerase II promoter and a modified splice sequence encoding the bovine auxin polynucleotides signal upstream of the 5' end of the viral genome. Contains the murine RNA polymerase I promoter between the two; contains the murine polymerase terminator I and polymerase terminator II sequences downstream of the 3' end of the viral genome; and is chimeric in the O-type foot-and-mouth disease virus O/CHA/ The core sequence of Hammerase and Hepatitis Ease at both ends of the 99 full-length cDNA genome, of which Hepatitis Ease has a total of 88 ribonucleic acids, and the self-cleavage modification site is at the 5' end G; the core sequence of Hammerase has a total of 58 A ribonucleic acid, the self-cleavage modification site is at the 3' terminal C, and the infectious clone containing the O-type foot-and-mouth disease virus O/CHA/99 genome is transfected into recipient cells, through the RNA polymerase II promoter and RNA The polymerase I promoter regulatory elements were respectively transcribed and packaged to produce viral RNA precursors, and then modified and cleaved by HamRz and HdvRz to produce infectious viral RNA. The plasmid prO/ CHA/99 and the partial L and P1 fragments of the A/WH/CHA/09 strain obtained above were digested with Afl II and SgrA I respectively, ligated and transformed into JM109 competent cells, and positive clones were identified by sequencing to obtain A/WH/CHA/09 strain partial leader protein L and structural protein P1 recombinant plasmid, the recombinant plasmid is named prA-FMDV, and only site 1 is different from the circulating strain. 5. a kind of preparation method of A type foot-and-mouth disease recombinant virus according to claim 4, is characterized in that: use described recombinant plasmid prA-FMDV, directly transfect the cell that foot-and-mouth disease virus is sensitive, preferred BHK-21 cell or IBRS- 2 cells to obtain the type A foot-and-mouth disease recombinant virus matching the antigen of the epidemic strain. 6. a vaccine prepared from the A-type foot-and-mouth disease recombinant virus according to claim 5 is characterized in that: after immunizing pigs and cattle, the body is effectively stimulated to produce an immune response, and the immune protection of pigs and cattle is provided, and the A-type AISA pedigree In the 10,000 times bovine half infectious dose (BID ₅₀ ) challenge experiment, the immune protection rate can reach 100%, and the 50% protective dose (PD ₅₀ ) is 10.81-13.59. 7. the application of a type A foot-and-mouth disease recombinant virus as claimed in claim 5 in preparing the vaccine for preventing the disease caused by type A foot-and-mouth disease virus, the vaccine is used for the prevention and control of type A foot-and-mouth disease virus in my country and its surrounding countries.

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