CN103266091A

CN103266091A - A type foot-and-mouth disease recombinant vaccine strains and preparation method and application thereof

Info

Publication number: CN103266091A
Application number: CN2013101753244A
Authority: CN
Inventors: 刘湘涛; 郑海学; 杨帆; 靳野; 郭建宏; 曹伟军; 张克山; �田宏; 何继军; 董海聚; 才学鹏
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2012-07-30
Filing date: 2013-05-14
Publication date: 2013-08-28
Anticipated expiration: 2033-05-14
Also published as: CN102757942A; CN103266090A; CN103266091B; CN103266090B

Abstract

The invention relates to A type foot-and-mouth disease recombinant vaccine strains prepared by using a reverse genetic manipulation technology and a preparation method and application of the strains. One strain is an A type foot-and-mouth disease recombinant vaccine strain with high titer, antigen matching property and immune protection rate, and the other strain is an A type foot-and-mouth disease recombinant non-pathogenic vaccine strain with high titer, antigen matching property and immune protection rate and without pathogenicity for a host; an antigen nucleotide sequence of each of the vaccine strains is shown as SEQ ID NO: 1; eukaryotic plasmids of viruses can be saved by using a reverse genetic manipulation system; after pigs and cattle are immunized by using the inactivated vaccines prepared from the prepared recombinant vaccine strains, the bodies can be effectively stimulated to produce immune response, and an immune protection effect is provided for the bodies of the pigs and the cattle; through a 10,000-times cattle median infectious dose (BID50) challenge assay of A type AISA topological strains, the immune protection rate reaches 100 percent, and the median protective dose (PD50) is 10.81 to 13.59; and the recombinant vaccine strains can be applied to prevention and control of A type foot-and-mouth disease viruses of China and neighboring countries.

Description

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

Technical field

The invention belongs to biotechnology and field of biological product; be specifically related to a kind of A type foot and mouth disease recombinant vaccine strain that utilizes the preparation of reverse genetic manipulation technology and its preparation method and application; utilize this method to prepare two kinds of vaccine strains; a kind of is the high A type foot and mouth disease recombinant vaccine strain of height, antigen matching and immune protective rate of tiring; another kind is that tire height, antigen matching, immune protective rate is high and to the A type foot and mouth disease reorganization no pathogenicity vaccine strain of host's no pathogenicity, can utilize above-mentioned two kinds of vaccine strains to produce two kinds of vaccines simultaneously.

Background technology

(Foot-and-mouth disease is by FMD virus (Foot-and-mouth disease virus, a kind of acute, hot, height contagious disease that artiodactyls such as the pig that FMDV) causes, ox and sheep infect FMD) to foot and mouth disease.FMDV is a kind of sub-thread positive chain RNA virus, belongs to Picornaviridae (Picornaviridae), Hostis (Aphthovirus).This virus is divided into 7 serotypes (A, O, C, Asial, SAT1, SAT2 and SAT3 type), and wherein A type foot and mouth disease virus is popular comparatively extensive, is the main epidemic isolates in the world that is only second to O type foot and mouth disease.In recent years, the foot and mouth disease epidemic situation frequently takes place and is the popular situation of globalization, this not only to world's livestock product economic structure huge threat, and caused the worry of people to social public health problems such as food safeties, cause country and regional Livestock Product Trade that epidemic situation takes place to suffer heavy losses.

A type foot and mouth disease is many in China surrounding countries generation and popular, and China found A type foot and mouth disease in 1958 first in area, Aketao, Xinjiang.Import area, China Inner Mongol into and cause fairly large popular from Mongolia at the beginning of 1963, epidemic situation involves more than 200 counties and cities of China 7 provinces, and after this nearly 40 in the period of, no A type foot and mouth disease epidemic situation is popular, yet 2009 the beginning of the year China Wuhan, area, Shanghai broken out A type foot and mouth disease epidemic situation in succession, and propagating into a plurality of provinces very soon, these cause of diseases belong to ASIA topological type strain.In February, 2013, pig A type foot and mouth disease is broken out in Maoming, Guangdong.On April 25th, 2013, ox A type foot and mouth disease epidemic situation also takes place in Ta Jie village, Lhasa city Dazi County Ta Jie township.On May 2nd, 2013, ox A type foot and mouth disease epidemic situation takes place in Suo Keman village, Bai Shiairike town, Aksu Prefecture Awat County, Xinjiang.Analyze through national foot and mouth disease reference laboratory and world's foot and mouth disease reference laboratory, the cause of disease that the foot and mouth disease epidemic situation took place in 2013 all belongs to South East Asia 97 strains (SEA-97 strain), it is new incoming epidemic situation overseas, belong to the ASIA topological type together with strain in 2009, homology is about 92%, and is and lower with China historical strain homology.Breaking out of A type foot and mouth disease makes that the complicated epidemic prevention situation of China's script is severeer.

The reason that foot-and-mouth disease antigen variation strain produces is that the epitope variant amino acid sequence produces the multifarious result of antigen; epitope is the important substance basis of mutagenesis host immune response, and the quality of the epitope coupling of vaccine strain and epidemic isolates is determining the height of vaccine immunity protection ratio.Replication and the virus stability of the popular virus of A type foot and mouth disease are lower, the whole genome sequence analysis revealed, the amino acid of the VP1 of its 3 ' UTR Nucleotide and cell receptor combining site has variation, has amino acid to morph in this position and may mean strain replication and the antigenic change of strain.Evolutionary tree analysis revealed, the A type foot and mouth disease of breaking out be inferior country southeast, with the no hereditary derivation relation of the popular poison of the domestic A type that once occurred.Vaccine inoculation is one of main means of prevention and control foot and mouth disease, the new vaccine kind poison of screening is a kind of method the most common, fast from popular poison, but not that each popular poison can both be tamed and become desirable vaccine kind poison, immunogenicity (spectrotype coupling, the induced animal body produces enough immunizing power behind the immune animal) or production performance (receive the poison time, produce poison amount and virus stability) are undesirable often.

Desirable vaccine kind poison is hard-earned, innovation vaccine kind poison triage techniques, and the permanent mechanism of setting up the renewal of vaccine kind poison is very urgent.The maturation of foot and mouth disease reverse genetic manipulation technology provides the novel method that solves the screening of vaccine kind poison for us, utilize it to transform and to modify virogene, artificial screening obtains the strain of expection biological characteristics, mate rapidly by replacing P1 gene and popular malicious antigen, obtain good vaccine strain and be prepared into vaccine to be used for the prevention and control epidemic isolates.

Summary of the invention

The objective of the invention is to solve the technical problem of the A type aftosa vaccine strain that current shortage antigen mates, production performance is bad, a kind of A type foot and mouth disease recombinant vaccine strain and its preparation method and application is provided, prepare vaccine with this vaccine strain deactivation, immune cattle is after 28 days, with 10000 times of BID ₅₀The toxic agent amount is attacked the poison experiment, observes continuously 12 days, and the result shows: full dosage 100% protection.50% protection dosage (PD ₅₀) 10.81～13.59, this vaccine can be used for prevention and the control of China and the A of surrounding countries type foot and mouth disease virus thereof; A kind of A type foot and mouth disease reorganization no pathogenicity vaccine strain and its preparation method and application also is provided simultaneously, and this vaccine strain can induce host's early immune to reply to pig and the equal no pathogenicity of ox, does not form toxaemia and toxin expelling not, has improved the biological safety of vaccine strain.

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

A kind of A type foot and mouth disease recombinant vaccine strain is characterized in that: described A type foot and mouth disease recombinant vaccine strain have high titre, with the antigen matching height of popular virus, the feature that immune protective rate is high, the antigen nucleotides sequence of this vaccine strain is classified as shown in the SEQ ID NO:1.

A kind of A type foot and mouth disease reorganization no pathogenicity vaccine strain; it is characterized in that: on the basis of above-mentioned A type foot and mouth disease recombinant vaccine strain, the L gene is suddenlyd change; described A type foot and mouth disease reorganization no pathogenicity vaccine strain have high titre, with the antigen matching height of popular virus; the immune protective rate height and to pig and ox no pathogenicity, do not form toxaemia, toxin expelling not; and can induce the host to produce early immune and feature such as reply, the L gene order of this vaccine strain is shown in the SEQ ID NO:2.

A kind of preparation method of described A type foot and mouth disease recombinant vaccine strain, it is characterized in that: the structure of (1), A type foot and mouth disease recombinant vaccine strain eukaryotic transcription plasmid, this eukaryotic transcription plasmid is embedded with hammer hammer enzyme (hammerhead ribozyme in viral full-length cDNA both sides, HamRz) and hepatitis E enzyme (hepatitis delta ribozyme, HdvRz), in 5 of its outside ' and hold to be added with polysaccharase I and polymerase II transcripting promoter, at 3 ' end terminator is arranged; System is skeleton with the rescue of O type foot and mouth disease virus, and by AflII and SgrAI restriction enzyme, the sequence SEQ ID NO:1 that contains partial L and P1 gene with the A/WH/CHA/09 strain replaces corresponding gene, obtains the recombinant plasmid of prA-FMDV; Used A/WH/CHA/09 strain is to be located away from Wuhan, Hubei in January, 2009, go down to posterity through BHK-21 and to cultivate for 10 generations, be preserved in animal doctor office of the Ministry of Agriculture and specify the preservation unit: national foot and mouth disease reference laboratory, use RNAeasy Mini Kit to extract total RNA of A/WH/CHA/09 strain, with the synthetic viral first chain cDNA of primer oligNot I reverse transcription, be template with the first synthetic chain cDNA, with the gene fragment of primer AP1-F and AP1-R amplification acquisition A/WH/CHA/09 strain partial L and P1, above-mentioned three Auele Specific Primers at the A/WH/CHA/09 strain are respectively:

oligNot?I：5`-ttttctaga gcggccgct ₃₈-3′

AP1-F：5′-ttttc cttaagggacaggaacatgctgtgtttgcctgcgt-3′

AP1-R：5′-tatttt caccggtgcaataattttctgcttgtgtctgt?c-3′，

In above Auele Specific Primer, contain the AflII restriction enzyme site among amplification A/WH/CHA/09 strain partial L and the used upstream primer AP1-F of P1 gene order; The SgrAI restriction enzyme site that contains the underscore part among the downstream primer AP1-R, increase with above-mentioned Auele Specific Primer, obtain A/WH/CHA/09 strain partial L and P1 gene fragment, size is 2400bp, make up 50 μ L reaction systems, amplification condition is: 94 ℃ of 5min, 94 ℃ of 30s, 57 ℃ of 30s, 68 ℃ of 2min30s, go to2,35 circulations, 72 ℃ of 8min, pcr amplification product carry out purifying and reclaim and deliver order-checking after 0.8% agarose gel electrophoresis is examined, obtain the DNA of said gene fragment;

PrO/CHA/99 is the plasmid that contains O/CHA/99 strain full-length cDNA that this laboratory makes up in earlier stage, wherein, the O/CHA/99 strain is O type foot and mouth disease strain, be located away from the Hong-Kong in 1999, now be stored in national foot and mouth disease reference laboratory, it is that sequence is sheared in the modification that contains human cytomegalic inclusion disease virus rna plymerase ii promotor and coding Polisac poly thuja acid signal in viral genome 5 ' end upstream that the prO/CHA/99 plasmid is formed, and contains mouse source rna plymerase i promotor between the two at it; Contain mouse source polysaccharase terminator I and polysaccharase terminator II sequence in viral genome 3 ' end downstream; And be entrenched in the hammer hammer enzyme at O type foot and mouth disease virus O/CHA/99 full-length cDNA genome two ends and the core sequence of hepatitis E enzyme, and wherein the hepatitis E enzyme has 88 Yeast Nucleic Acid, and the oneself shears decorating site at 5 ' terminal G place; Hammer hammer enzyme core sequence has 58 Yeast Nucleic Acid, the oneself shears decorating site at 3 ' terminal C place, to contain the genomic infections clone transfection of O type foot and mouth disease virus O/CHA/99 to recipient cell, transcribe respectively by rna plymerase ii promotor and rna plymerase i promoter regulation element and to pack out the viral RNA precursor, shear to produce through the modification of HamRz and HdvRz again and have infective viral RNA, plasmid prO/CHA/99 and the above-mentioned A/WH/CHA/09 strain partial L that obtains and P1 fragment with O type foot and mouth disease virus O/CHA/99 strain rescue system, after using AflII and SgrAI double digestion respectively, connect and be converted in the JM109 competent cell, positive colony is identified in order-checking, acquisition contains the recombinant plasmid of A/WH/CHA/09 strain part leader protein L and structural protein P1, with recombinant plasmid called after prA-FMDV, and only site 1 is variant with epidemic isolates;

(2), A type foot and mouth disease recombinant plasmid prA-FMDV transfection foot and mouth disease virus sensitive cells, be specially BHK-21 cell or IBRS-2 cell, rescue obtains A type foot and mouth disease recombinant virus described and epidemic isolates antigen coupling; This eukaryotic transcription plasmid all can be saved out corresponding virus in adapting to cell, suckling mouse and pig body.

The preparation method of described A type foot and mouth disease recombinant vaccine strain, it is characterized in that: with described recombinant plasmid prA-FMDV direct transfection foot and mouth disease virus sensitive cells BHK-21 cell or IBRS-2 cell, obtain the A type foot and mouth disease recombinant virus with epidemic isolates antigen coupling.

A kind of preparation method of described A type foot and mouth disease reorganization no pathogenicity vaccine strain, it is characterized in that: the structure of (1), A type foot and mouth disease reorganization no pathogenicity vaccine strain eukaryotic transcription plasmid, on the basis of above-mentioned recombinant plasmid prA-FMDV, with L gene SAP zone (SAF-A/B, Acinus, and PIAS) sudden change, its building process is: expression plasmid pCDNA3.1 (+) is increased with Auele Specific Primer, obtain containing the gene fragment of PacI and AflII restriction enzyme site, above-mentioned primer is respectively:

pCD-AflII-ApaI-F：5′-ttttc cttaaggggcccgtttaaacccgctgat-3′

pCD-PacI-NheI-R：5′-cttac ttaattaagctagccagcttgggtctcccta-3′

Simultaneously with recombinant plasmid prA-FMDV, behind PacI and AflII double digestion, be connected with the above-mentioned pCDNA gene fragment that contains PacI and AflII restriction enzyme site that obtains and be converted in the JM109 competent cell, positive colony is identified in order-checking, acquisition contains the recombinant plasmid of 5 ' UTR and partial L gene, with recombinant plasmid called after pCD-OL; To identify that correct recombinant plasmid pCD-OL is template, carry out pcr amplification with phosphorylation mutant primer F-P and SAP-P, the point mutation primer of above-mentioned 5 ' phosphorylation is respectively:

F-P：5′-gacctcacagggcttgaactgcacga-3′

SAP-P：5′-ctccgcctgcttggcggctgcaagcgtg-3′，

In the point mutation primer of above phosphorylation, SAP zone in the L gene is suddenlyd change, with this primer is put down terminal amplification, purifying reclaims the PCR product, after room temperature connects 5min, transformed into escherichia coli JM109, the positive colony after the point mutation is identified in order-checking, with the rite-directed mutagenesis recombinant plasmid called after pCD-OL-SAP that obtains;

With the recombinant plasmid pCD-OL-SAP after the SAP zone suddenlys change in acquired recombinant plasmid prA-FMDV and the above-mentioned L gene that obtains, after using PacI and AflII double digestion respectively, connect and be converted in the JM109 competent cell, positive colony is identified in order-checking, acquisition contains the recombinant plasmid of SAP zone sudden change in the P1 gene of A/WH/CHA/09 strain and the L gene, with recombinant plasmid called after prA-SAP-FMDV;

(2), A type foot and mouth disease reorganization no pathogenicity vaccine strain eukaryotic transcription plasmid prA-SAP-FMDV transfection foot and mouth disease virus sensitive cells, BHK-21 cell or IBRS-2 cell, rescue obtain A type foot and mouth disease recombinant virus described and epidemic isolates antigen coupling and no pathogenicity.

The preparation method of described A type foot and mouth disease reorganization no pathogenicity vaccine strain, it is characterized in that: on the basis of recombinant plasmid prA-FMDV, SAP to the L gene suddenlys change, cell BHK-21 cell or IBRS-2 cell with recombinant plasmid prA-SAP-FMDV direct transfection foot and mouth disease virus sensitivity, obtaining the pig of recombinant virus rA-SAP-FMDV and ox does not all have pathogenic, and do not form toxaemia, toxin expelling can not produce strong and immunne response early.

The vaccine that a kind of described A type foot and mouth disease recombinant vaccine strain is made; it is characterized in that: the A type foot and mouth disease recombinant vaccine strain of cultivation divinyl imines (Binary ethylenimine; BEI) deactivation; concentrated and purified back is mixed with vaccine with the ISA206 adjuvant with 1: 1 volume ratio; the effective stimulus body produces immunne response behind immune swine and the ox; and pig and ox body immanoprotection action are provided, to 10000 times of ox 50 3nfective dose (BID of A type AISA pedigree strain ₅₀) attack the poison experiment, immune protective rate can reach 100%, median protective dose (PD ₅₀) be 10.81～13.59.

The application in the vaccine of the disease that preparation prevention A type foot and mouth disease virus causes of a kind of described A type foot and mouth disease recombinant vaccine strain and A type foot and mouth disease reorganization no pathogenicity vaccine strain, this vaccine can be used for prevention and the control of China and the A of surrounding countries type foot and mouth disease virus thereof.

The present invention has following positively effect:

China's foot and mouth disease A type Wuhan poison popular proposed new research topic to the screening of deactivation seed poison with changing.This virocyte pathology time instability, titre are low, do not reach production requirement.The vaccine kind of current international practice poison screening principle is at present: the one, and spectrotype is checked the number, and can effectively protect the current popular strain; The 2nd, immunogenicity is strong, but induced animal produces enough strong immune response; The 3rd, production performance is good, the vaccine candidate strain have the pathology time short, titre is high and pathology production performance preferably such as stable, can be used for suitability for industrialized production.Screening new vaccine strain from popular seed culture of viruses is a kind of method the most common and that can take effect fast, has directly solved the problem that spectrotype is checked the number.But to two other problem, immunogenicity and production performance problem still need to test to prove.And reality is, is not that each epidemic isolates can both be tamed into desirable vaccine kind poison, and immunogenicity or production performance are undesirable often.Show that by a large amount of tests desirable vaccine kind is difficultly malicious, innovation vaccine kind poison screening (structure) technology, the permanent mechanism of setting up the renewal of vaccine kind poison is very urgent.

The maturation of foot and mouth disease reverse genetic technology provides the novel method of the screening that solves vaccine kind poison for us.Realize transformation and modification to virogene by the reverse genetic technology, can manually obtain to expect the strain of biological characteristics, and can join type with epidemic isolates, in the hope of epidemic isolates mates with making a variation rapidly.Can reduce epidemic isolates domestication link as far as possible and bring negative impact.Establish solid basis for preparing efficient vaccine later on fast, it is significant that integral body is improved the FMD vaccine quality.The present invention is exactly by the efficient reversed genetic system of having set up, transformation by genes involved, setting up the strain that the pathology time is short, virus titer is high, solve production and the domestication problem of screening vaccine kind poison, serves as to make up efficient vaccine kind poison to lay the foundation with this vaccine strain framework.

According to the foot and mouth disease molecular epidemiology, utilize reverse genetics to carry out the multiple phenotype of FMD vaccine strain and improve.

1), improved the production performance (as virus titer and pathology time) of FMD vaccine strain in viral integral level, the strain of the present invention's preparation can improve titre more than 100 times, has reduced the antigen prepd cost.Recombinant virus rA-FMDV is replaced and saved out to epidemic isolates A/WH/CHA/09 partial L and P1 gene, measure the viral biology feature, the result shows, this recombinant virus rA-FMDV has the advantages that the pathology time is short, titre is high, recombinant virus rA-FMDV is in the 5th generation; the pathology time is reduced to 11h, is stabilized in about 10h from the complete pathology time in 9 generation the 5th generation to the.And popular malicious A/WH/CHA/09 is in the 6th generation in generation to 12, and after 13 generations, the pathology time just shortens about 10h about 25～20h the pathology time fully.Virus titer (the TCID of rA-FMDV ₅₀) reach 6.8 in the 5th generation, from 9 generation of the 5th generation to the virus titer be stabilized in more than 7.5, and popular poison the 5th generation titre be 2.8, from 9 generation of the 5th generation to the virus titer surely between 2.8 to 4.5.This shows that recombinant virus has significantly improved its production performance.

2), realize to improve vaccine strain antigen matching and immunologic responsiveness, the structural protein of the vaccine strain that the present invention makes up are consistent with epidemic isolates, antigen mates fully, has improved the specific aim of vaccine strain and epidemic isolates;

3), the vaccine strain of the present invention preparation contains potential vaccine sign site, for the later development marker vaccine lays the foundation;

4), compare with the vaccine of epidemic isolates preparation, the vaccine strain of the present invention's preparation has improved vaccine immune response and protectiveness height, PD ₅₀Value has strengthened vaccine field prevention and control effect 10.81～13.59;

5), the pig of reorganization no pathogenicity vaccine strain rA-SAP-FMDV of the present invention and ox all do not have pathogenicly, and do not form toxaemia, toxin expelling can not produce early stage immunne response.

6) and according to surrounding countries' epidemic situation, by the technology of the present invention, can establish the storehouse of vaccine strategic reserves targetedly, realize need not the vaccine strain building mode of epidemic isolates (the motionless poison of living);

7), the technology of the present invention changed vaccine strain screening acclimatization technology be subjected to viral natural character restriction big, waste time and energy, defective that success ratio is low, can realize that more initiatively effective vaccine strain makes up, realized foot and mouth disease inactivated vaccine seed culture of viruses preparation technology's innovation, having major application is worth, promote vaccine seed culture of viruses technical matters, belong to the leading level in the world.

Description of drawings

Fig. 1 is the electrophorogram of foot and mouth disease virus A/WH/CHA/09 strain partial L and P1 gene fragment in the example 1.

Fig. 2 is the foot and mouth disease virus rescue prO/CHA/99 of system in the example 2.

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

Fig. 4 is example 2 recombinant strains and epidemic isolates site 1 difference comparison diagram.

Fig. 5 infects the CPE that causes behind the BHK-21 cell for rescue recombinant virus rA-FMDV, rA-SAP-FMDV in the example 3.

Fig. 6 is for having inoculated the BHK-21 cellmediated immunity fluorogram of rescue recombinant virus rA-FMDV in the example 4.1.

Fig. 7 is rescue recombinant virus rA-FMDV and the virulence test comparison diagram of epidemic isolates A/WH/CHA/09 on the BHK-21 cell in the example 5.2.

Embodiment

The present invention in conjunction with national foot and mouth disease reference laboratory in recent years strain accumulation and to the analysis and research of its whole genome sequence, part leader protein L and structural protein P1 gene with China A type foot and mouth disease strain A/WH/CHA/09, replace by restriction endonuclease sites and by corresponding nucleotide sequence in the rescue system of the O type foot and mouth disease virus O/CHA/99 strain of setting up, obtain a kind of A type foot and mouth disease virus recombinant plasmid prA-FMDV; Described specificity nucleotide sequence is shown in SEQ ID NO:1.

A kind of reverse genetic operating system for preparing A type foot and mouth disease recombinant vaccine strain, this system comprises: (1) eukaryotic transcription plasmid, this transcribes plasmid can express described reorganization A type foot and mouth disease virus full-length gene cDNA sequence, plasmid is embedded with ribozyme (hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz)) in viral full-length cDNA both sides, in 5 of its outside ' hold to be added with polysaccharase (polymerase, pol) I and pol II transcripting promoter have terminator at 3 ' end; (2) A type foot and mouth disease recombinant plasmid prA-FMDV transfection sensitive cells, preferred BHK-21 cell or IBRS-2 cell.Described A type foot and mouth disease recombinant virus obtains with the rescue of above-mentioned rescue system, and this plasmid can be saved out corresponding virus in adapting to cell, suckling mouse and pig body.

The infections clone of A type foot and mouth disease recombinant virus, be that rescue system with the strain of O type foot and mouth disease virus vaccine is skeleton, by AflII and SgrAI restriction enzyme, contain partial L and P1 gene replacement corresponding gene with the A/WH/CHA/09 strain, obtain the recombinant plasmid of prA-FMDV.

A kind of preparation method of A type foot and mouth disease recombinant virus, use described recombinant plasmid, the cell of direct transfection foot and mouth disease virus sensitivity, preferred BHK-21 cell or IBRS-2 cell, obtain the A type foot and mouth disease recombinant virus with epidemic isolates antigen coupling, the A type foot and mouth disease recombinant virus of this method preparation, called after rA-FMDV.

The construction process of above-mentioned A type foot and mouth disease virus recombinant plasmid prA-FMDV is to adopt Auele Specific Primer that the genome of A type foot and mouth disease strain A/WH/CHA/09 is increased, and obtains part leader protein L and structural protein P1 gene.Utilize special AflII and SgrAI restriction enzyme site that the plasmid prO/CHA/99 of O type foot and mouth disease virus is carried out the gene replacement, make up and obtain A type foot and mouth disease virus recombinant plasmid called after prA-FMDV.Cytopathogenic effect (CPE) appears in transfection BHK-21 cell the 2nd generation 48h, and continuous passage has all detected the FMDV of rescue by RT-PCR, indirect immunofluorescence, reverse indirect hemagglutination and the experiment of suckling mouse virulence etc.After biological characteristics is measured, compare recombinant virus rA-FMDV with popular poison and have pathology time weak point, the characteristics that virus titer is high.

In the application of above-mentioned A type foot and mouth disease recombinant viral vaccine strain, the genetically engineered virus rA-FMDV that will be obtained by the sex clone of A type foot and mouth disease recombinant virus infection, vaccine is made in deactivation, measures the PD of this vaccine ₅₀Between 10.81～13.59.Solve the difficult problem in the actual production, obtained the A type foot and mouth disease efficient vaccine strain of improvement.By attacking the band poison condition detection behind the poison, the morbidity ox can detect the band poison, and the immunoprotection ox does not form and is with malicious phenomenon.Comprehensive These parameters, this recombinant strain is an efficient vaccine strain.

A kind of recombinant plasmid preparation method of A type foot and mouth disease reorganization no pathogenicity, utilize specific phosphorylation primer that point mutation is carried out in the SAP zone of the L gene of A type foot and mouth disease virus recombinant plasmid prA-FMDV, utilize PacI and AflII restriction enzyme site to replace the corresponding gene of the recombinant plasmid prA-FMDV that has made up, structure obtains the A type foot and mouth disease virus recombinant plasmid of SAP zone sudden change, called after prA-SAP-FMDV.Cytopathogenic effect (CPE) appears in transfection BHK-21 cell the 5th generation 48h, and continuous passage has all detected the FMDV of rescue by RT-PCR, indirect immunofluorescence, reverse indirect hemagglutination and the experiment of suckling mouse virulence etc.After biological characteristics is measured, compare recombinant virus rA-SAP-FMDV with popular poison and have characteristics to host pig and the equal no pathogenicity of ox.

Below in conjunction with concrete embodiment the present invention is done description further, but concrete enforcement do not done any restriction to the present invention.

Employed experimental technique is normal condition if no special instructions in the following example, as " fine works molecular biology experiment guide " (F.M. Ao Sibai, R.E. James Kingston, graceful chief editor, the Ma Xuejun of waiting of J.G. Saden, the Su Yuelong translates, Beijing: Science Press, 2004) described in method carry out.

The acquisition of embodiment 1.A type foot and mouth disease virus A/WH/CHA/09 strain partial L and P1 gene order.

The used A/WH/CHA/09 strain of the inventor is by the preservation of the designated state man of animal doctor office of Ministry of Agriculture foot and mouth disease reference laboratory, and the public can obtain by the trust letter of animal doctor office of Ministry of Agriculture written instructions.Use RNAeasy Mini Kit (Qiagen company) to extract total RNA of A/WH/CHA/09 strain, with the synthetic viral first chain cDNA of primer oligNot I reverse transcription, be template with the first synthetic chain cDNA, obtain the gene order of A/WH/CHA/09 strain with primer AP1-F and AP1-R amplification.Above-mentioned three Auele Specific Primers at the A/WH/CHA/09 strain are respectively oligNot I (5`-ttttctaga Gcggccgct ₃₈-3 '), AP1-F (5 '-ttttc CttaagGgacaggaacatgctgtgtttgcctgcgt-3 ') and AP1-R (5 '-tatttt CaccggtgCaataattttctgcttgtgtctgt c-3 ').In above Auele Specific Primer, contain the AflII restriction enzyme site among amplification A/WH/CHA/09 strain partial L and the used upstream primer AP1-F of P1 gene order; Contain SgrAI restriction enzyme site (underscore part) among the downstream primer AP1-R.Increase with above-mentioned Auele Specific Primer, obtain A/WH/CHA/09 strain partial L and P1 gene fragment, size is 2400bp (Fig. 1), conforms to the expection size.The Premix LA of used suitable long segment amplification, the excellent property of increasing

(TaKaRa company) archaeal dna polymerase, make up 50 μ L reaction systems according to product description, amplification condition is: 94 ℃ of 5min, 94 ℃ of 30s, 57 ℃ of 30s, 68 ℃ of 2min30s, go to2,35 circulations, 72 ℃ of 8min, pcr amplification product carries out purifying and reclaims and deliver order-checking after 0.8% agarose gel electrophoresis is examined, obtain the DNA of said gene fragment; The electrophoresis result of amplified production as shown in Figure 1.

The structure of embodiment 2.A type foot and mouth disease recombinant virus infection sex clone.

On the frame foundation of the O type foot and mouth disease virus O/CHA/99 strain rescue prO/CHA/99 of system that has set up, O type foot and mouth disease strain is saved the prO/CHA/99 of system as shown in Figure 2: contain human cytomegalic inclusion disease virus rna plymerase ii promotor (Human cytomegalovirus RNA polymerase II promoter, P in viral genome 5` end upstream _II) and the modification of coding Polisac poly thuja acid signal shear sequence, contain mouse source rna plymerase i promotor (Mouse RNA polymerase I promoter, P between the two at it _I); Contain mouse source polysaccharase terminator I (Murine terminator I, T in viral genome 3` end downstream _I) and polysaccharase terminator II (Murine terminator II, T _II) sequence; And the hammer that is entrenched in O type foot and mouth disease virus O/CHA/99 full-length cDNA genome two ends is hammered enzyme (Hammerhead ribozyme into shape, HamRz) and hepatitis E enzyme (Hepatitis delta ribozyme, HdvRz) core sequence, wherein the hepatitis E enzyme has 88 Yeast Nucleic Acid, and the oneself shears decorating site at the terminal G of 5` place; Hammer hammer enzyme core sequence has 58 Yeast Nucleic Acid, and the oneself shears decorating site at the terminal C of 3` place.To contain the genomic infections clone transfection of O type foot and mouth disease virus O/CHA/99 to recipient cell, transcribe respectively by rna plymerase ii promotor and rna plymerase i promoter regulation element and to pack out the viral RNA precursor, shear to produce through the modification of HamRz and HdvRz again and have infective viral RNA.

After the plasmid prO/CHA/99 of O type foot and mouth disease virus O/CHA/99 strain rescue system and partial L that example 1 obtains and P1 fragment used AflII and SgrAI double digestion respectively, connect and be converted in the JM109 competent cell, positive colony is identified in order-checking, acquisition contains the recombinant plasmid of A/WH/CHA/09 strain part leader protein L and structural protein P1, with recombinant plasmid called after prA-FMDV (Fig. 3), and only site 1 and epidemic isolates variant (Fig. 4).

A type foot and mouth disease reorganization no pathogenicity recombinant plasmid preparation method:

A type foot and mouth disease recombinant virus L gene SAP zone (SAF-A/B, Acinus, and PIAS) sudden change, its infections clone building process is: expression plasmid pCDNA3.1 (+) is increased with Auele Specific Primer, obtain containing the gene fragment of PacI and AflII restriction enzyme site, above-mentioned primer is respectively:

pCD-AflII-ApaI-F：5′-ttttc cttaaggggcccgtttaaacccgctgat-3′

pCD-PacI-NheI-R：5′-cttac ttaattaagctagccagcttgggtctcccta-3′

F-P：5′-gacctcacagggcttgaactgcacga-3′

SAP-P：5′-ctccgcctgcttggcggctgcaagcgtg-3′，

The rescue of embodiment 3.A type foot and mouth disease recombinant virus.

Produce with QIAGEN company The recombinant plasmid prA-FMDV and the prA-SAP-FMDV that contain the full genome cDNA of A type foot and mouth disease recombinant virus that the preparation of Plus Maxi Kit purifying is obtained by example 2.Treat that BHK-21 monolayer growth to 80%～90% o'clock is used for transfection.Clean cell with the OPTI-MEMI substratum, at liposome Lipofectamine ^TMWith the recombinant plasmid transfection BHK-21 cell of 4 μ g, it is cultivated in containing 37 ℃ of incubators of 5%CO2 under the mediation of 2000 (Invitrogen companies), set up liposome contrast and normal cell contrast simultaneously.4～6h inhales and removes cell conditioned medium after the transfection, adds MEM substratum (Invitrogen company), continues to cultivate the appearance situation of observation of cell pathology; Results virus when treating that 90% left and right sides pathology appears in cell.Inoculate the BHK-21 cell again behind the multigelation 3 times, can stably produce CPE up to virus, cell rounding occurs, become botryoidalis to distribute, the final cell disintegration fragmentates (as shown in Figure 5).With A type foot and mouth disease recombinant virus called after rA-FMDV; Recombinant virus called after rA-SAP-FMDV with the sudden change of A type foot and mouth disease virus L gene SAP zone.In Fig. 4, A: the picture that infects the CPE of BHK-21 cell appearance for rescue recombinant virus rA-FMDV; B: be normal control BHK-21 cell picture.C: the picture that infects the CPE of BHK-21 cell appearance for rescue recombinant virus rA-SAP-FMDV

The evaluation of embodiment 4. rescue A type foot and mouth disease recombinant viruses.

4.1 indirect immunofluorescence detects virus antigen.

With the BHK-21 cell in 2 generations of blind passage to the after the transfection, behind-70 ℃ of multigelations three times, be seeded to the growth of having placed slide glass in the bottom by a certain percentage and have in six orifice plates of BHK-21 cell (monolayer cell grows to 60%～70%), place 37 ℃ of incubators that contain 5%CO2, do indirect immunofluorescence behind the 24h according to a conventional method, primary antibodie is A type FMDV rabbit positive serum, and two anti-are flag F ITC goat anti-rabbit igg (Sigma company), establish the normal cell contrast simultaneously.Inoculate the 2nd generation enchylema the BHK-21 cell in the visible green specificity fluorescent, and the no visible fluorescence of normal cell contrast produces (A as shown in Figure 6: for having inoculated the BHK-21 cell of saving recombinant virus rA-FMDV; B: be the normal BHK-21 cell of virus inoculation not), show the expression that FMDV albumen is arranged in the BHK-21 cell of recombinant virus infection.

4.2 reverse indirect hemagglutination qualification test

Collect 1ml in BHK-21 cell the 7th generation rescue virus that time that CPE occurs tends towards stability that goes down to posterity, 58 ℃ of 40min deactivations, with the virus after the deactivation by doing serial dilution (establishing the positive control of blank and popular malicious A/WH/CHA/09 simultaneously) at 1: 2, each extent of dilution is got 50 μ L and is added in 96 orifice plates, the sheep red blood cell (SRBC) that adds 25 μ L FMDV antibody sensitizedizations again, put the 1～2min that vibrates on the micro mixer, add a cover, observations behind the room temperature placement 2h.When the FMDV antibody on being attached at hemocyte meets with the antigen that dissociates, form antigen-antibody aggegation network, sheep red blood cell (SRBC) is aggegation thereupon also, macroscopic hemagglutination phenomenon occurs, thereby according to cell deposition situation result of determination.

The result shows, when pressing the rA-FMDV infection BHK-21 cell of dilution in 1: 32 red cell agglutination takes place, and the red corpuscle deposition do not occur; Red cell agglutination appears in popular malicious 75% of dilution in 1: 32, and the negative control sample red corpuscle occurs and deposits fully; Reverse indirect hemagglutination assay is the result show: cause the cytopathic FMDV of being of BHK-21.

The virulence test of embodiment 5. rescue A type foot and mouth disease recombinant viruses.

5.1 the suckling mouse virulence is tested.

To reach rescue virus and the popular poison in the 9th generation respectively at the BHK-21 cell and use 10 times of doubling dilutions of PBS damping fluid respectively, respectively get 10 ^-5～10 ^-9The viral liquid of doubling dilution is through subcutaneous vaccination 2～4 age in days suckling mouses, and each extent of dilution is respectively inoculated 4, and dosage of inoculation is 200 μ L/, observes 8d continuously.4 suckling mouse inoculations of blank group PBS damping fluid, 200 μ L/ only observe and record the death condition of suckling mouse, calculate LD with the Karber method ₅₀The suckling mouse of inoculation rescue virus and popular poison behind inoculation 20h, part show expiratory dyspnea, after acroparalysia and press from both sides symptoms such as the coda sound is hoarse with tweezers.The suckling mouse of the popular poison of inoculation and rescue recombinant virus is dead successively behind the 48h, and the control group suckling mouse is normal.According to statistics, calculate the LD of the viral rA-FMDV of rescue at last ₅₀Be 10 ^-7.5/ 0.2mL, the LD of rA-SAP-FMDV ₅₀Be 10 ^-6.5/ 0.2mL, the LD of epidemic isolates A/WH/CHA/09 ₅₀10 ^-5.0/ 0.2mL.

5.2 the test of the virulence on the BHK-21 cell.

According to ordinary method the BHK-21 cell is digested, add the MEM cell culture medium contain 10% foetal calf serum, be sub-packed in 28 2mL Tissue Culture Flasks, in 37 ℃ of incubators that contain 5%CO2, cultivate, treat that cell monolayer length is standby by 90% o'clock.With MEM with 10 times of doubling dilutions virus liquid, with each extent of dilution (10 ^-3.0～10 ^-8.0) viral liquid add the 2mL Tissue Culture Flask respectively, 4 bottles of each extent of dilution, connect the back 37 ℃ of senses of poison by 10% and make 1h, clean cell 2～3 times with the MEM that does not contain serum, add the MEM that does not contain serum, put into 37 ℃ of incubators that contain 5%CO2 and cultivate, observed 3 days, with the median infective dose (TCID of Read-MuenchShi method mensuration to the BHK-21 cell ₅₀).Measure the titre in the viral rA-FMDV of rescue and parent's poison 1-9 generation according to this method, and statistics each for the pathology time.

After saving viral rA-FMDV inoculation BHK-21 cell, in 5 generations of passage to the, the pathology time is reduced to 11h, is stabilized in about 10 h from the complete pathology time in 9 generation the 5th generation to the; And parent's poison is in the 6th generation in generation to 12, and after 13 generations, the pathology time just shortens about 10 h about 25-20h the pathology time fully.Carry out TCID with this poison ₅₀The test, calculate with the Read-MuenchShi method, the genetically engineered poison of transformation is in the 5th generation, titre reaches 6.8, from 9 generation of the 5th generation to the virus titer be stabilized in more than 7.5.Parent's poison the 5th generation titre be 2.8, from 9 generation of the 5th generation to the virus titer surely between 2.8 to 4.5.Save viral rA-SAP-FMDV and go down to posterity stable back mensuration virus titer more than 7.17.

5.3 the virulence of recombinant virus rA-SAP-FMDV test.

BHK-21 is gone down to posterity with the MEM cultivation that contains 10% foetal calf serum.Recombinant virus rA-SAP-FMDV is saved in the preparation of rolling bottle individual layer BHK-21 passage cell culture method routinely, and the viral liquid of results is viral cultures, places-40 ℃ of preservations, and the reorganization poison adopts injecting pathway to attack poison, and attacking the toxic agent amount is 10 ⁷TCID ₅₀, pig muscle injection 2ml/ head, the subcutaneous injection 2ml/ of cow tongue portion head is set up epidemic isolates A/WH/CHA/09 control group simultaneously, observes continuously 10 days, and take temperature changes and the observed and recorded incidence.Above-mentioned experiment with pig and ox all available from Pest-or disease-free area, and through the FMD liquid phase blocking-up ELISA (LPB-ELISA) that national foot and mouth disease reference laboratory is produced detect A type antibody all＜1: 4.FMD Nonstructural Protein 3ABC-ELISA antibody test is negative.Attack malicious result and show, rA-SAP-FMDV attacks poison back pig and clinical symptom does not all appear in ox, namely pig and ox is not all had pathogenicly, sees Table 1.

Table 1 recombinant strain and epidemic isolates A/WH/CHA/09 virulence test clinical symptom

The no clinical symptom of-expression

5.4 the pig of recombinant virus rA-SAP-FMDV and ox produce early stage immunne response.

Prepare the rA-SAP-FMDV recombinant virus according to the method described above, respectively pig is carried out intramuscular injection 2ml/ head (5 * 10 ⁷TCID ₅₀), ox is carried out tongue subcutaneous injection 2ml/ head (5 * 10 ⁷TCID ₅₀), and gather separation of serum every day in inoculation back, and detecting A type antibody with FMD liquid phase blocking-up ELISA (LPB-ELISA), antibody horizontal the results are shown in Table 2 all greater than 1: 45 in the time of the 3rd day.

Table 2rAsia1-RSD-SAP-FMDV recombinant virus is attacked poison back antibody horizontal change list to the pig ox

Preparation and the safety check of embodiment 6. rescue A type foot and mouth disease recombinant viral vaccines.

BHK-21 is gone down to posterity with the DMEM cultivation that contains 10% foetal calf serum.Rolling bottle individual layer BHK-21 passage cell culture method prepares rA-FMDV and saves viral liquid routinely, and the viral liquid of results is viral cultures, places-40 ℃ of preservations.(Binary ethylenimine, BEI) (Sigma company) 26 ℃ of deactivations add BEI one time, again deactivation 24h again behind the 24h with 3mmol/l divinyl imines with the viral cultures deactivation.Safety check after the deactivation, concentrated and purified with antigen, antigen cutting and ISA206 adjuvant (French SEPPIC) are prepared vaccine with 1: 1 mixed.Specifically the foot and mouth disease inactivated vaccine rules according to veterinary biologics in the Pharmacopoeia of the People's Republic of China prepare.The subcutaneous injection inactivation of viruses 2ml/ of safety check cow tongue portion head was observed 6 continuously day by day, the observation period ox in good health, hoof and mouth nose are all no abnormal.

Above-mentioned experiment be available from Pest-or disease-free area with ox, and through the FMD liquid phase blocking-up ELISA (LPB-ELISA) that national foot and mouth disease reference laboratory is produced detect the O type and AsiaI type antibody equal＜1: 4.FMD Nonstructural Protein 3ABC-ELISA antibody test is negative.

The comparison of embodiment 7. vaccine immunity animal effects.

7.1 vaccine immunity is attacked malicious protection test

16 oxen of A type foot and mouth disease recombinant viral vaccine immunity that resulting safety check in above-described embodiment 6 is qualified, establishing 2 non-immune cattle contrasts simultaneously (requires to be not less than 1: 6 by the foot and mouth disease titre against the enemy of the ox of immunity, it is negative to infect antibody), measure its immune efficacy.Attack malicious method and as a result decision method all according to described in " Manual of diagnostic tests and vaccines for terrestrial animals " (version in 2009, OIE (OIE)).Behind the immune cattle 28 days, with 10000 times of ID ₅₀The toxic agent amount is attacked the poison experiment; observed 12 days continuously; the result shows: with this A type foot and mouth disease recombinant virus as vaccine strain; the BHK-21 cell propagation back deactivation of cultivating; make vaccine with ISA206 adjuvant (French SEPPIC) emulsification; immune cattle can effectively be protected the attack of the popular poison of Chinese current A type foot and mouth disease.Result such as table 3.

Table 3A type foot and mouth disease recombinant viral vaccine immune cattle is attacked poison back clinical symptom and protection situation

7.2 in the cross immunity and the experiment.

The A type foot and mouth disease resulting serum of recombinant viral vaccine immune animal and rA-FMDV that resulting safety check in above-described embodiment 6 is qualified, A/WH/CHA/09 and O/CHA/99 three strain poison carry out in the viral cross immunity and experiment, measure antibody matching value r, wherein 1 〉=r 〉=0.3 shows that strain and vaccine have matched, can resist the attack of this strain behind the vaccine immunity animal, can be used as potential vaccine strain; R≤0.3 shows that strain and vaccine matching are relatively poor, the attack that can not resist corresponding strain behind the vaccine immunity animal.Result such as table 4.

The viral cross immunity neutralization test of table 4. rA-FMDV, the antibody matching value of A/WH/CHA/09 and O/CHA/99

7.3 vaccine immunity is renderd a service experiment (PD ₅₀)

Test with ox available from Pest-or disease-free area, and through the FMD liquid phase blocking-up ELISA (LPB-ELISA) that national foot and mouth disease reference laboratory is produced detect A type antibody all＜1: 4.FMD Nonstructural Protein 3ABC-ELISA antibody test is negative.It is all strict with the stable breeding of ABSL-3 laboratory that this tests used animal.72 experiments are divided into 4 groups with ox, and control group is the vaccine that popular malicious A/WH/CHA/09 preparation is crossed in immunity, and experimental group is the vaccine that the rA-FMDV poison preparation of recombinating is crossed in immunity, and attacking poison is the popular malicious A/WH/CHA/09 of 10,000 times of BID50 with poison.Attack malicious mode and divide the multi-point injection venom for the lingual surface intracutaneous.Observed 30 continuously, occur foot and mouth disease symptoms such as bubble, ulcer according to cow tongue face, gums, hoof and judge that viral liquid causes the ox incidence, thereby judge that the cell venom is to the virulence situation of ox.The immune efficacy measurement result shows that the PD50 of recombinant strain is 10.81～13.59, and the vaccine PD50 of epidemic isolates preparation is at 5.57 (tables 5).

Table 5 recombinant strain and epidemic isolates PD ₅₀The experiment comparison sheet

Embodiment 8. vaccine immunity lead for animals poison detects.

Utilize two kinds of methods of Nonstructural Protein 3ABC antibody ELISA and viral nucleic acid real-time quantitative PCR to monitor, its amplifying nucleic acid real-time quantitative adopts TaqMan PCR method technology, according to foot and mouth disease virus 5 '-UTR district internal ribosome entry site conserved sequence designs synthetic primer SA-IR-219-246F:5`-CACYTYAAGRTGACAYTGRTACTGGTAC-3; SA-IR-315-293R:5`-CAGATYCCRAGTGWCICITGTTA-3` and probe SAmulti2-P-IR-292-269R:5`-CCTCGGGGTACCTGAAGGGCATCC-3` are used for fast, responsive, the immune band poison situation of attacking the back 32 days blood of poison, OP liquid of specific detection.The result shows that the morbidity animal can produce 3ABC antibody, and can monitor viral nucleic acid.And the immunoprotection animal can not monitor antibody and nucleic acid (table 6,7)

Table 6 Nonstructural Protein 3ABC antibody ELISA detects the antibody result in the serum

Dpc: the fate behind the poison is attacked in the expression immunity

+ be expressed as positive;-expression negative sample

Table 7 viral nucleic acid real-time quantitative PCR detected result

+ be expressed as positive;-expression negative sample

The fate behind the poison is attacked in the numeral immunity, the numeral ct value (the ct value is less than 35 positive samples) in the bracket

Above-described embodiment has only explained embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, also can make other improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

The present invention relates to a kind of efficient reversed genetic technique, prepare the high A type foot and mouth disease recombinant vaccine strain of height, antigen matching and immune protective rate of tiring with this technology; And the A type foot and mouth disease reorganization no pathogenicity vaccine strain to host's no pathogenicity is prepared in further improvement; Relate to the methods and applications of producing vaccine with the vaccine strain of preparation simultaneously.Described rescue system is the artificial constructed efficient eucaryon plasmid that can express accurate foot-and-mouth disease virus genome RNA, can make up, improves and prepare the foot and mouth disease recombinant virus whereby.Use above-mentioned plasmid can prepare high titre and the good vaccine strain of antigen matching; A type foot and mouth disease recombinant vaccine strain is prepared into deactivation vaccine; but the effective stimulus body produces immunne response behind immune swine and the ox; and pig and ox body immanoprotection action are provided, to 10000 times of ox 50 3nfective dose (BID of A type AISA topological type strain ₅₀) attack the poison experiment, immune protective rate reaches 100%, 50% protection dosage (PD ₅₀) be 10.81～13.59, can be used for prevention and the control of China and the A of surrounding countries type foot and mouth disease virus thereof.A type foot and mouth disease reorganization no pathogenicity vaccine strain is to host's no pathogenicity, does not copy in host, toxin expelling not, and the vaccine strain that can induce host's early immune to reply has improved biological safety and the immunne response ability of vaccine strain.

SEQ?ID?NO:1

CTTAAGGGCAGGAACACGCAGTGTTTGCCTGTGTTACCTCCAACGGGTGGTACGCGATTGACGACGAGGACTTTTACCCCTGGACACCAGACCCGTCTGATGTCCTGGTGTTTGTCCCGTATGATCAAGAACCACTCAACGGAGAATGGAAAACAAAGGTTCAGAGGCGACTTAAAGGGGCAGGGCAATCTAGCCCCGCCACCGGGTCGCAGAACCAGTCAGGCAATACCGGCAGCATCATTAACAACTACTACATGCAGCAGTACCAGAACTCCATGGACACACAACTTGGTGACAACGCCATCAGCGGAGGATCCAACGAGGGGTCCACGGACACAACCTCTACCCACACAACCAACACCCAAAACAATGACTGGTTCTCAAAACTGGCAAGTTCTGCATTCACCGGTCTTTTCGGCGCACTGCTCGCCGACAAGAAGACCGAAGAGACAACTCTTCTGGAGGACCGTATCCTCACCACTCGTAATGGACACACCACCTCTACAACTCAGTCGAGTGTGGGGGTCACCTACGGGTATTCAACTGGTGAGGACCACGTTTCTTGGACCTAACACATCAGGTTTGGAGACGCGGGTGGTACAAGCTGAAAGGTTCTTCAAGAAGCACTTGTTTGATTGGACAACGGACAAACCCTTTGGTCACATTGAAAAGCTGGAACTTCCCACTGATCACAAAGGTGTCTACGGACAGCTGGTGGACTCCTTTGCATACATGAGAAATGGCTGGGACGTGGAGGTGTCTGCTGTTGGCAACCAGTTCAACGGCGGGTGCCTTCTCGTGGCCATGGTACCTGAGTTTAAGGAGTTCACCACACGTGAAAAGTACCAGCTCACCCTGTTCCCCCACCAGTTCATTAGCCCCAGAACCAACATGACCGCGCACATCACGGTCCCGTACCTTGGTGTGAACAGGTATGACCAGTACAACAAACACAAACCCTGGACGTTGGTGGTGATGGTGGTTTCGCCACTTACCACTAGCTCCATTGGTGCATCACAGATTAAGGTCTACACCAACATCGCCCCGACCCACGTTCACGTGGCTGGCGAGCTCCCGTCGAAAGAGGGGATCGTGCCAGTCGCCTGCTCGGACGGGTACGGTGGCCTGGTGACAACAGACCCTAAAACAGCTGACCCTGCTTACGGTATGGTGTACAACCCACCTAGGACCAACTACCCCGGGCGGTTTACAAACTTGTTGGACGTGGCAGAGGCGTGCCCCACCTTCCTCTGTTTCGACGACGGGAAACCGTACGTTGTGACAAGAACGGACGAGCAGCGCCTCTTGGCCAAGTTTGACCTTTCCCTTGCTGCAAAGCACATGTCAAACACCTACCTTTCAGGGATAGCACAGTACTACGCACAGTACTCTGGCACCATCAATTTGCACTTCATGTTTACTGGTTCCACTGACTCAAAGGCCCGTTACATGGTGGCTTACGTCCCGCCCGGCGTGACAACGCCACCGGACACGCCTGAGAGAGCTGCGCACTGCATCCACGCAGAATGGGACACGGGGCTAAACTCCAAATTCACTTTTTCAATCCCATACGTATCTGCTGCAGATTACGCGTACACAGCGTCCGATGTGGCAGACACAACAAACGTACAGGGATGGGTTTGCATCTACCAAATCACCCATGGGAAGGCCGAACAAGACACTCTGGTTGTGTCGGTCAGCGCCGGCAAAGACTTTGAGCTGCGCCTCCCCATTGACCCCCGTGCGCAAACCACCGCCACCGGGGAATCAGCAGACCCCGTCACAACCACCGTCGAGAACTACGGTGGTGAGACACAAGTGCAGCGACGCCACCACACCGACGTCAGCTTCATAATGGACAGGTTTGTGCAAATCAAGCCTGTGAGCCCCACACATGTCATTGACCTCATGCAAACACACCAACACGGGCTGGTGGGCGCTATGTTGCGCGCGGCCACCTACTACTTTTCTGATCTTGAGATTGTGGTGAACCACACGGGTCGCCTAACGTGGGTACCCAATGGAGCACCTGAGGCAGCACTGGACAACACGAGCAACCCCACTGCTTACCACAAAGCACCGTTCACACGGCTTGCACTCCCTTACACCGCGCCACACCGCGTGTTGGCAACTGTGTACAACGGGAATAGCAAGTACTCTGCGCCTGCAACACGGCGAGGTGACTTGGGGTCTCTCGCGGCGAGGCTCGCCGCACAGCTTCCTGCCTCCTTCAACTACGGCGCGATTCGAGCCACGGAGATCCAAGAACTCCTCGTGCGCATGAAGCGTGCCGAGCTCTACTGCCCCAGGCCACTGCTGGCGGTGGAGGTGACGTCACAAGACAGACACAAGCAGAAAATTATTGCACCGGTG

SEQ?ID?NO:2

ATGAATACAACTGACTGTTTTATCGCTTTGGTACAGGCTATCAGAGAGATTAAAGCACTTTTCCTACCACGCACCACAGGAAAAATGGAACTGACACTGTACAACGGTGAGAAGAAGACCTTTTACTCCAGGCCCAACAACCACGACAACTGCTGGTTGAACGCCATCCTCCAGTTGTYCAGGTACGTTGAAGAACCATTCTTCGACTGGGTCTACAGTTCGCCTGAGAACCTCACGCTTGCAGCCGCCAAGCAGGCGGAGGACCTCACAGGGCTTGAACTGCACGAGGGTGGACCACCTGCTCTCGTGATCTGGAACATCAAGCACTTGCTCCACACCGGCATTGGCACCGCCTCGCGACCCAGCGAGGTGTGCATGGTGGATGGTACGGACATGTGCTTGGCTGATTTCCATGCAGGCATTTTCCTTAAGGGGCAAGAACACGCTGTGTTCGCGTGTGTCACCTCCAACGGGTGGTACGCGATTGATGATGAGGACTTCTACCCCTGGACGCCGGACCCATCCGACGTTCTGGTGTTTGTCCCGTACGATCAAGAACCACTCAACGGGGAATGGAAAGCCAAGGTTCAACGCAAGCTCAAA

Claims

1. A type foot and mouth disease recombinant vaccine strain; it is characterized in that: described A type foot and mouth disease recombinant vaccine strain have high titre, with the antigen matching height of popular virus; the feature that immune protective rate is high, the antigen nucleotides sequence of this vaccine strain is classified as shown in the SEQ ID NO:1.

2. A type foot and mouth disease reorganization no pathogenicity vaccine strain; it is characterized in that: on the basis of above-mentioned A type foot and mouth disease recombinant vaccine strain, the L gene is suddenlyd change; described A type foot and mouth disease reorganization no pathogenicity vaccine strain have high titre, with the antigen matching height of popular virus; the immune protective rate height and to pig and ox no pathogenicity, do not form toxaemia, toxin expelling not; and can induce the host to produce early immune and feature such as reply, the L gene order of this vaccine strain is shown in the SEQ ID NO:2.

3. the preparation method of an A type foot and mouth disease recombinant vaccine strain as claimed in claim 1, it is characterized in that: the structure of (1), A type foot and mouth disease recombinant vaccine strain eukaryotic transcription plasmid, this eukaryotic transcription plasmid is embedded with hammer hammer enzyme (hammerhead ribozyme in viral full-length cDNA both sides, HamRz) and hepatitis E enzyme (hepatitis delta ribozyme, HdvRz), be added with polysaccharase I and polymerase II transcripting promoter at the 5' in its outside end, at the 3' end terminator arranged; System is skeleton with the rescue of O type foot and mouth disease virus, by AflII and SgrAThe I restriction enzyme, the sequence SEQ ID NO:1 that contains partial L and P1 gene with the A/WH/CHA/09 strain replaces corresponding gene, obtains the recombinant plasmid of prA-FMDV; Used A/WH/CHA/09 strain is to be located away from Wuhan, Hubei in January, 2009, go down to posterity through BHK-21 and to cultivate for 10 generations, be preserved in animal doctor office of the Ministry of Agriculture and specify the preservation unit: national foot and mouth disease reference laboratory, use RNAeasy Mini Kit to extract total RNA of A/WH/CHA/09 strain, use primer olig NotThe viral first chain cDNA is synthesized in the I reverse transcription, is template with the first synthetic chain cDNA, and with the gene fragment of primer AP1-F and AP1-R amplification acquisition A/WH/CHA/09 strain partial L and P1, above-mentioned three Auele Specific Primers at the A/WH/CHA/09 strain are respectively:

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

AP1-F:5'-ttttc cttaagggacaggaacatgctgtgtttgcctgcgt-3'

AP1-R:?5'-tatttt caccggtgcaataattttctgcttgtgtctgt?c-3'，

In above Auele Specific Primer, contain among amplification A/WH/CHA/09 strain partial L and the used upstream primer AP1-F of P1 gene order AflThe II restriction enzyme site; Contain underscore part among the downstream primer AP1-R SgrAThe I restriction enzyme site increases with above-mentioned Auele Specific Primer, obtains A/WH/CHA/09 strain partial L and P1 gene fragment, size is 2400 bp, makes up 50 μ L reaction systems, and amplification condition is: 94 ℃ of 5min, 94 ℃ of 30s, 57 ℃ of 30s, 68 ℃ of 2min30s, go to 2,35 circulations, 72 ℃ of 8min, pcr amplification product carry out purifying and reclaim and deliver order-checking after 0.8% agarose gel electrophoresis is examined, obtain the DNA of said gene fragment;

PrO/CHA/99 is the plasmid that contains O/CHA/99 strain full-length cDNA that this laboratory makes up in earlier stage, wherein, the O/CHA/99 strain is O type foot and mouth disease strain, be located away from the Hong-Kong in 1999, now be stored in national foot and mouth disease reference laboratory, it is that sequence is sheared in the modification that contains human cytomegalic inclusion disease virus rna plymerase ii promotor and coding Polisac poly thuja acid signal in viral genome 5' end upstream that the prO/CHA/99 plasmid is formed, and contains mouse source rna plymerase i promotor between the two at it; Contain mouse source polysaccharase terminator I and polysaccharase terminator II sequence in viral genome 3' end downstream; And be entrenched in the hammer hammer enzyme at O type foot and mouth disease virus O/CHA/99 full-length cDNA genome two ends and the core sequence of hepatitis E enzyme, and wherein the hepatitis E enzyme has 88 Yeast Nucleic Acid, and the oneself shears decorating site at the terminal G of 5' place; Hammer hammer enzyme core sequence has 58 Yeast Nucleic Acid, the oneself shears decorating site at the terminal C of 3' place, to contain the genomic infections clone transfection of O type foot and mouth disease virus O/CHA/99 to recipient cell, transcribe respectively by rna plymerase ii promotor and rna plymerase i promoter regulation element and to pack out the viral RNA precursor, shear to produce through the modification of HamRz and HdvRz again and have infective viral RNA, with plasmid prO/CHA/99 and the above-mentioned A/WH/CHA/09 strain partial L that obtains and the P1 fragment of O type foot and mouth disease virus O/CHA/99 strain rescue system, use respectively AflII and SgrABehind the I double digestion, connection is converted in the JM109 competent cell, and positive colony is identified in order-checking, obtains to contain the recombinant plasmid of A/WH/CHA/09 strain part leader protein L and structural protein P1, with recombinant plasmid called after prA-FMDV, and only site 1 is variant with epidemic isolates;

4. the preparation method of A type foot and mouth disease recombinant vaccine strain according to claim 3, it is characterized in that: with described recombinant plasmid prA-FMDV direct transfection foot and mouth disease virus sensitive cells BHK-21 cell or IBRS-2 cell, obtain the A type foot and mouth disease recombinant virus with epidemic isolates antigen coupling.

5. the preparation method of an A type foot and mouth disease as claimed in claim 2 reorganization no pathogenicity vaccine strain, it is characterized in that: the structure of (1), A type foot and mouth disease reorganization no pathogenicity vaccine strain eukaryotic transcription plasmid, on the basis of above-mentioned recombinant plasmid prA-FMDV, with L gene SAP zone (SAF-A/B, Acinus, and PIAS) sudden change, its building process is: expression plasmid pCDNA3.1 (+) is increased with Auele Specific Primer, contained PacI and AflThe gene fragment of II restriction enzyme site, above-mentioned primer is respectively:

pCD- AflII- ApaI-F：5'-ttttc cttaaggggcccgtttaaacccgctgat-3'

pCD- PacI- NheI-R：5'-cttac ttaattaagctagccagcttgggtctcccta-3'

Simultaneously with recombinant plasmid prA-FMDV, warp PacI and AflBehind the II double digestion, with above-mentioned containing of obtaining PacI and AflThe pCDNA gene fragment connection of II restriction enzyme site is converted in the JM109 competent cell, and positive colony is identified in order-checking, obtains to contain the recombinant plasmid of 5 ' UTR and partial L gene, with recombinant plasmid called after pCD-OL; To identify that correct recombinant plasmid pCD-OL is template, carry out pcr amplification with phosphorylation mutant primer F-P and SAP-P, the point mutation primer of above-mentioned 5' phosphorylation is respectively:

F-P：5'-gacctcacagggcttgaactgcacga-3'

SAP-P：5'-ctccgcctgcttggcggctgcaagcgtg-3',

Recombinant plasmid pCD-OL-SAP with after the SAP zone suddenlys change in acquired recombinant plasmid prA-FMDV and the above-mentioned L gene that obtains uses respectively PacI and AflBehind the II double digestion, connect and to be converted in the JM109 competent cell, positive colony is identified in order-checking, obtains to contain the recombinant plasmid of SAP zone sudden change in the P1 gene of A/WH/CHA/09 strain and the L gene, with recombinant plasmid called after prA-SAP-FMDV;

6. the preparation method of A type foot and mouth disease according to claim 5 reorganization no pathogenicity vaccine strain, it is characterized in that: on the basis of recombinant plasmid prA-FMDV, SAP to the L gene suddenlys change, cell BHK-21 cell or IBRS-2 cell with recombinant plasmid prA-SAP-FMDV direct transfection foot and mouth disease virus sensitivity, obtaining the pig of recombinant virus rA-SAP-FMDV and ox does not all have pathogenic, and do not form toxaemia, toxin expelling can not produce strong and immunne response early.

7. vaccine that A type foot and mouth disease recombinant vaccine strain according to claim 1 is made; it is characterized in that: the A type foot and mouth disease recombinant vaccine strain of cultivation divinyl imines (Binary ethylenimine; BEI) deactivation; concentrated and purified back is mixed with vaccine with the ISA206 adjuvant with the 1:1 volume ratio; the effective stimulus body produces immunne response behind immune swine and the ox; and pig and ox body immanoprotection action are provided, to 10000 times of ox 50 3nfective dose (BID of A type AISA pedigree strain ₅₀) attack the poison experiment, immune protective rate can reach 100%, median protective dose (PD ₅₀) be 10.81～13.59.