CN111961654B - Heat-resistant phenotype-stable genetic recombinant foot-and-mouth disease virus (FMDV) avirulent strain carrying negative marker and O/A type FMDV bivalent inactivated vaccine - Google Patents

Abstract

The invention discloses a heat-resistant phenotype stable inheritance recombinant foot-and-mouth disease virus avirulent strain carrying negative markers and an O/A type foot-and-mouth disease bivalent inactivated vaccine. The FMDV virus cDNA infectious clone plasmid is constructed, carries a virus capsid heat-resistant phenotype molecular determinant factor, a molecular factor losing replication capacity in vivo, a negative marker factor of 3A and 3B protein epitope deletion and Pst I enzyme cutting sites introduced at two sides of a P1 coding region, can be quickly constructed by replacing the capsid protein coding region thereof with any epidemic strain, saves a heat-stable and marked foot-and-mouth disease virus avirulent strain, and is used as a foot-and-mouth disease inactivated vaccine seed virus. The universal plasmid is used for constructing two recombinant viruses aiming at the current two dominant epidemic strains and preparing an O/A type foot-and-mouth disease bivalent inactivated vaccine, and immune animals induce high-level neutralizing antibodies and generate immune protection; the antibody detection of the vaccinated animal can realize the differential diagnosis of the vaccinated animal and the naturally infected animal.

Description

Heat-resistant phenotype-stable genetic recombinant foot-and-mouth disease virus (FMDV) avirulent strain carrying negative marker and O/A type FMDV bivalent inactivated vaccine

Technical Field

The invention relates to a virus full-length cDNA infectious clone plasmid and a recombinant virus strain obtained by rescuing the full-length cDNA infectious clone plasmid, in particular to an FMDV full-length cDNA infectious clone plasmid and a heat-resistant phenotype-stable genetic recombinant foot-and-mouth disease virus avirulent strain which is obtained by rescuing by adopting a reverse genetic technology and carries a 3B epitope negative marker.

Background

Foot-and-Mouth Disease (FMD) is caused by Foot-and-Mouth Disease Virus (FMDV), and mainly damages an acute, febrile, highly contact infectious Disease of artiodactyl animals such as pigs, cattle and sheep (Grubman and Baxt.2004.clinical micro. Rev.17: 465-. The disease is epidemic in many countries of asia, africa and the middle east, the financial burden of epidemic prevention, the loss caused by outbreak of disease and the influence of international trade restriction on the economy and society of epidemic countries are all huge, and the introduction of disease into epidemic-free countries often leads to catastrophic consequences. Considering the high infectivity of FMD, the serious harm it poses to epidemic countries, and the enormous threat to non-epidemic countries, it is internationally known as political economic disease, which has been highly valued by governments throughout, OIE has listed it as the first infectious disease in a class of animals.

FMDV belongs to the family of picornaviridae, members of the genus foot-and-mouth disease virus, and has seven serotypes (A, O, C, Asia1, SAT1, SAT2, and SAT3) and several tens of subtypes, with no immunological cross-protection between strains of each serotype and only partial cross-protection between strains of each subtype. The viral genome is a single-stranded positive-strand RNA, has a total length of about 8.5kb, and consists of a 5' noncoding region (5' UTR), an Open Reading Frame (ORF), and a 3' UTR, the ORFs of which encode 4 structural proteins (VP4, VP2, VP3, and VP1) and 8 nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C, and 3D).

The immunization of the inactivated vaccine is an effective means for preventing and controlling foot and mouth disease in epidemic countries, and the ring-shaped immunization is also commonly adopted in the outbreak of epidemic-free countries to reduce the loss caused by the overlarge slaughter quantity of animals. However, there are four main problems in the production and use of inactivated foot-and-mouth disease vaccine, and there is a need to solve the following problems: firstly, the virulent strain is used as the seed virus for producing the inactivated vaccine, so that the risk of virus dispersion caused by factory leakage exists, and serious consequences are brought once the virus is generated; secondly, the virus is mutated or new epidemic strains are introduced, so that the original immune barrier can be broken through to cause immune failure, the seed virus for producing the vaccine needs to be replaced in time, and the replacement is limited and time-consuming for virulent strains; third, the foot and mouth disease virus 146S capsid is susceptible to disaggregation, thereby affecting the immunoprotective efficacy of the vaccine and the shelf life of the vaccine, and methods for increasing heat resistance by artificial mutation have been reported to improve the stability of the foot and mouth disease virus capsid (ahay kotech et al, nature structural & molecular biology,2015), but the heat resistant phenotype is unstable (Katherine a. scott et al, Journal of Virology, 2016); fourthly, in the process of vaccine preparation, the non-structural proteins such as 3A, 3B and the like of the foot-and-mouth disease virus are difficult to clean, so that the non-structural proteins are remained in the vaccine, and the non-structural proteins can induce the generation of specific antibodies after the animals are subjected to multiple immunizations, so that the vaccinated animals and naturally infected animals are difficult to distinguish by using a differential diagnosis technology taking the detection of the non-structural proteins as a reference, and the implementation of a foot-and-mouth disease immune control plan and the quarantine in live animal trade are difficult to solve.

Research on attenuation of FMDV, with great effort in many countries around the world over the last 60 years, has not been successful despite major advances. The FMDV attenuation studies, whether using traditional passaging adaptation techniques or modern genetic engineering techniques, have met an insurmountable hurdle: the resulting attenuated phenotype is limited by the animal species and no effective method or approach for attenuating all three major artiodactyl pigs, cattle or sheep could be found. The existing foot-and-mouth disease virus genetic engineering attenuation research mainly focuses on the non-structural proteins L, 3A and 3D of the virus, but the problem cannot be solved.

Disclosure of Invention

One of the purposes of the invention is to provide a universal platform for FMDV cDNA infectious clone plasmids and a recombinant foot-and-mouth disease virus avirulent strain which is constructed and rescued by adopting the reverse genetic operation platform, does not replicate in susceptible animals and has 3A and 3B protein epitope negative markers;

the second purpose of the invention is to provide an O-type FMDV full-length cDNA infectious clone plasmid and an O-type recombinant FMDV non-virulent strain which is obtained by rescuing the cDNA infectious clone plasmid by adopting a reverse genetic technology, has stable heredity of heat-resistant phenotype, does not replicate in a susceptible animal body and has 3A and 3B protein epitope negative markers;

the third purpose of the invention is to provide an A-type FMDV full-length cDNA infectious clone plasmid and an A-type recombinant FMDV non-virulent strain which is obtained by rescuing the cDNA infectious clone plasmid by adopting a reverse genetic technology, has stable heredity of heat-resistant phenotype, does not replicate in a susceptible animal body and has 3A and 3B protein epitope negative markers;

the fourth purpose of the invention is to provide a bivalent inactivated vaccine for preventing and treating O/A type foot-and-mouth disease.

The above object of the present invention is achieved by the following technical solutions:

the invention firstly provides FMDV full-length cDNA infectious clone plasmid, wherein a structural domain 4 of IRES of FMDV genome is replaced by a structural domain 4 of IRES of bovine rhinovirus, a coding region of 84-143 amino acids of FMDV virus non-structural protein 3A is deleted, a coding region of 3B3 protein of FMDV is used for replacing coding regions of 3B1 protein and 3B2 protein of FMDV, and Pst I enzyme digestion sites are introduced at two sides of the FMDV structural protein P1 coding region.

In order to maintain the genetic stability of the virus and prevent the two 3B3 coding sequences from homologous recombination, the coding sequence of the 3B3 protein used for replacing 3B1 and 3B2 is subjected to codon optimization, and the optimized nucleotide sequence is shown as SEQ ID NO. 1.

As a preferred embodiment of the invention, the nucleotide sequence of the FMDV full-length cDNA infectious clone plasmid is shown as SEQ ID NO. 2.

The invention further utilizes FMDV full-length cDNA infectious clone plasmid to obtain a recombinant foot-and-mouth disease virus (FMDV) avirulent strain through rescue by a reverse genetic method, wherein the recombinant FMDV avirulent strain carries a heat-resistant capsid, does not replicate in a susceptible animal body and has the characteristics of 3A and 3B epitope negative markers.

The method for obtaining the recombinant foot-and-mouth disease virus avirulent strain by rescuing the FMDV full-length cDNA infectious clone plasmid by a reverse genetic method comprises the following steps: after linearization, the FMDV full-length cDNA infectious clone plasmid is in vitro transcribed to obtain the genome RNA of the recombinant virus, and the recombinant FMDV is rescued by transfected cells; wherein the cell may be a BHK-21 cell.

Further, the invention provides a full-length cDNA infectious cloning plasmid of the FMDV, wherein a structural protein P1 coding region of the full-length cDNA infectious cloning plasmid of the FMDV is replaced by a structural protein P1 coding region of the FMDV; wherein, the 79 th tyrosine (Y) of the structural protein VP2 of the O-type FMDV is mutated into histidine (H) and the 93 th serine (S) is mutated into tyrosine (Y).

As a preferred embodiment, the nucleotide sequence of the O-type FMDV infectious full-length cDNA clone plasmid is shown as SEQ ID NO. 3.

The invention further utilizes the O-type FMDV full-length cDNA infectious clone plasmid to rescue an O-type recombinant FMDV virus-free strain by adopting a reverse genetic method, wherein the O-type recombinant FMDV virus-free strain has genetically stable heat-resistant phenotype, does not replicate in susceptible animals and has the characteristics of 3A and 3B epitope negative markers.

The method for obtaining the recombinant O-type FMDV virus-free strain by rescuing the full-length cDNA infectious clone plasmid of the O-type FMDV by using a reverse genetic method comprises the following steps: after linearization, the full-length cDNA infectious clone plasmid of the O-type FMDV is subjected to in vitro transcription to obtain genome RNA of a recombinant virus, and a transfected cell saves the recombinant O-type FMDV; wherein the cell may be a BHK-21 cell.

Further, the invention provides a full-length cDNA infectious cloning plasmid of the A-type FMDV, wherein a structural protein P1 coding region of the full-length cDNA infectious cloning plasmid of the FMDV is replaced by a structural protein P1 coding region of the A-type FMDV; wherein alanine (A) at position 3 is mutated to threonine (T) and asparagine (N) at position 17 is mutated to histidine (H) in the structural protein VP1 of FMDV type A.

As a preferred embodiment, the nucleotide sequence of the full-length cDNA infectious clone plasmid of the A-type FMDV is shown as SEQ ID NO. 4.

The invention further utilizes the full-length cDNA infectious clone plasmid of the A-type FMDV to rescue and obtain an A-type recombinant FMDV virus-free strain, and the A-type recombinant FMDV virus-free strain has genetically stable heat-resistant phenotype, does not replicate in susceptible animals and has the characteristics of 3A and 3B epitope negative markers.

The method for obtaining the recombinant A-type FMDV virus-free strain by rescuing the full-length cDNA infectious clone plasmid of the A-type FMDV by using a reverse genetic method comprises the following steps: after linearization, the full-length cDNA infectious clone plasmid of the A-type FMDV is subjected to in vitro transcription to obtain the genome RNA of the recombinant virus, and the transfected cells save the recombinant A-type FMDV; wherein the cell may be a BHK-21 cell.

The invention also provides a method for constructing the recombinant O-type foot-and-mouth disease virus mutant strain with stable inheritance of capsid heat-resistant phenotype, which comprises the steps of mutating the 79 th amino acid of the structural protein VP2 of O-type FMDV from tyrosine (Y) to histidine (H) and mutating the 93 th amino acid from serine (S) to tyrosine (Y); the capsid heat-resistant phenotype of the recombinant O type FMDV mutant strain constructed by the method can be stably inherited.

The invention further provides a method for constructing a recombinant FMDV mutant strain with stably inherited capsid heat-resistant phenotype, which comprises the steps of mutating the 3 rd amino acid of the structural protein VP1 of FMDV type A from alanine (A) to threonine (T) and mutating the 17 th amino acid from asparagine (N) to histidine (H); the capsid heat-resistant phenotype of the recombinant A-type FMDV mutant strain constructed by the method can also be stably inherited.

The invention further provides the use of the recombinant FMDV strain in the following aspects:

the recombinant FMDV non-virulent strain is applied to preparing a medicament for preventing and treating foot-and-mouth disease or an application in preparing a reagent for identifying and diagnosing a vaccinated animal and a naturally infected animal;

the recombinant O-type FMDV virus-free strain is applied to preparing a medicine for preventing and treating O-type foot-and-mouth disease or an application in preparing a reagent for identifying and diagnosing a vaccinated animal and a naturally infected animal;

the A-type recombinant FMDV non-virulent strain is applied to preparing a medicament for preventing and treating A-type foot-and-mouth disease or an application in preparing a reagent for identifying and diagnosing vaccinated animals and naturally infected animals;

simultaneously applying the recombinant O-type FMDV avirulent strain and the recombinant A-type FMDV avirulent strain to prepare a bivalent inactivated vaccine for preventing and treating O-type foot-and-mouth disease and A-type foot-and-mouth disease;

the bivalent inactivated vaccine of the present invention may further comprise a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient may be a functional molecule serving as a vehicle, an adjuvant, a carrier or a diluent. The pharmaceutically acceptable excipient may be a transfection facilitating agent, including surfactants such as Immune Stimulating Complexes (ISCOMS), Freunds incomplete adjuvant, LPS analogs (including monophosphoryl lipid a), muramyl peptides, quinone analogs, squalene (squalene), hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles, and the like;

the vaccines of the present invention can be administered by various routes including oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, by inhalation, by buccal administration, intrapleural, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, and intraarticular, or combinations thereof. For veterinary use, the vaccine may be prepared in an acceptable formulation for administration in accordance with normal veterinary practice. Veterinarians can readily determine the dosage regimen and route of administration that is most appropriate for a particular animal. The bivalent inactivated vaccine of the present invention may be administered using a conventional syringe, a needle-free injection device, or the like.

Summary of the invention

Previous studies by the present inventors found that replacement of FMDV IRES domain 4(IRES-Fd4) with IRES chimeric virus FMDV (R4) generated by Bovine Rhinovirus (BRBV) IRES domain 4(IRES-Bd4) at 10⁶TCID₅₀Head dose neck muscle inoculation natural host pig, does not express any foot and mouth disease clinicSymptoms, no viremia, no toxin expulsion from the oral and nasal cavities, and no antibody induction by the virus in pigs, which indicates that IRES chimeric virus FMDV (R4) loses replication ability in susceptible animals (CN108085302A, WO/2018/090994). To further evaluate the pathogenicity of FMDV (R4) in swine, the present invention used the most sensitive heel intradermal route, using a higher dose (10) of FMDV (R4) vaccination⁷TCID₅₀Head or 10⁸TCID₅₀Perhead) and inoculated in the same way 10⁵TCID₅₀The results of the ultra-high dose, most sensitive route vaccination test with the/head dose of wild-type virus O/YS/CHA/05 as a virulent control showed that FMDV (R4) does not replicate in pigs, is unable to establish an infection and thus is completely non-pathogenic to pigs.

In order to construct a molecular marker virus which can be used for differential diagnosis, the invention carries out comparative research on the molecular marker mode on the 3B protein; the comparison result shows that the method and the strategy are feasible for carrying out differential diagnosis on the vaccinated animals and the naturally infected animals by replacing the 3B1 protein and the 3B2 protein with the 3B3 protein (the nucleotide sequence of the optimized coding gene is shown in SEQ ID NO. 1) to remove the main antigenic region of the 3B protein so as to realize the molecular negative marker of the foot-and-mouth disease virus and then using the antibody detection ELISA method established by the monoclonal antibody 2H 1.

Abhay Kotech et al found that the S2093Y (amino acid S at position 93 of VP2 protein mutated to Y) mutation significantly increased the thermostability of FMDV capsids (Abhay Kotech et al, natural structural & molecular biology, 2015). Subsequently, Katherine A.Scott et al found that the S2093Y mutation was not stably inherited as a molecular determinant of the heat-resistant phenotype of SAT 2FMDV (Katherine A.Scott et al, Journal of Virology, 2016). In view of the above, the invention uses the full-length cDNA infectious clone plasmids pYS and pQSA of O-type and A-type FMDV to respectively construct and save the heat-resistant mutant strains rO/YS-S2093Y and rA/QSA-Q2093C of VP2 protein amino acid site-directed mutation, and finds that the heat-resistant phenotypes of the mutant strains are also unstable, the heat-resistant performance of the virus is gradually weakened in the process of in vitro passage, and the heat-resistant performance is completely lost when the virus is transmitted to the 5 th generation; sequencing of passage viruses of different generations shows that the mutant rO/YS-S2093Y has S2093Y mutated to S2093H when the mutant rA/QSA-Q2093C has the reversion mutation of Q2093C when the mutant rA/QSA-Q2093C is transmitted to the 5 th generation, thereby causing the heat-resistant phenotype of the mutant to disappear. The mutation studies of O-type and A-type FMDV of the present invention support the research results of Katherine A et al in SAT 2-type FMDV, further indicating that amino acid 93 of VP2 protein found by Abhay Kotecha et al as a determinant for thermotolerant phenotype molecules is not stably inherited.

In order to obtain genetically stable FMDV heat-resistant mutant strains, the invention carries out multiple rounds of hot pressure screening on mutant viruses rO/YS-S2093Y and rA/QSA-Q2093C, and finally obtains O type FMDV heat-resistant mutant strains rO/YS delta and A type FMDV heat-resistant mutant strains rA/QSA delta. The heat-resistant mutant strains rO/YS delta and rA/QSA delta obtained by heat-resistant pressure screening are respectively subjected to heat-resistant phenotype stability detection (action at 56 ℃ for 1h) in 10 generations of BHK-21 cells without pressure, and the heat resistance of the 10 th (P10) virus and the heat resistance of the primary (P0) virus of the two heat-resistant mutant strains are not changed, so that the heat-resistant phenotypes have genetic stability. Sequencing the structural protein coding region of the heat-resistant mutant showed that: the P10 generation virus of the O type FMDV heat-resistant mutant rO/YS delta only contains Y2079H and S2093Y 2 amino acid mutations like the P0 virus; like the P0 virus, the P10 generation virus of the heat-resistant mutant rA/QSA delta contains only 2 amino acid mutations of A1003T (the 3 rd amino acid A of VP1 protein is mutated into T) and N1017H (the 17 th amino acid N of VP1 protein is mutated into H), while Q2093C of the mutant virus has a back mutation. Cloning pYS and pQSA with O-type and A-type FMDV infectious cDNA, respectively, and performing single mutation and combined mutation on mutation sites of the heat-resistant mutant strains to determine molecular determinants for heat resistance of O-type and A-type FMDV. As a result, it was found that the Y2079H mutation alone did not increase the thermotolerance of type O FMDV, and no thermotolerance analysis was performed because the S2093Y mutation alone was not stably inherited; however, the Y2079H and S2093Y combined mutations significantly enhanced the heat tolerance properties of type O FMDV. Mutations a1003T or N1017H alone did not increase the thermotolerant characteristics of FMDV type a, while the combined mutations significantly enhanced the thermotolerant characteristics of FMDV type a. Thus, the present inventors have found that the Y2079H mutation, while not producing a thermotolerant phenotype, can be stably inherited in concert with the S2093Y thermotolerant mutation, thus synergistically determining the capsid thermotolerant stability phenotype of type O FMDV; the combined mutations of a1003T and N1017H synergistically determine the capsid thermostable phenotype of type a FMDV and are stably inherited.

Based on the research results of the inventor, the invention further constructs a universal plasmid pIRES with 3A and 3B epitope negative markers and capsid heat-resistant phenotype based on FMDV infectious clone plasmid with IRES domain 4 replaced_Bd43AB_mPst I, a recombinant FMDV avirulent strain, carrying a molecular marker and stable capsid thermotolerant phenotype can be created for any circulating strain.

In order to construct FMDV non-virulent strain with distinguishable diagnostic marker as foot-and-mouth disease inactivated vaccine seed virus, the present invention performs the following genetic engineering operation based on infectious cDNA clone plasmid pFMDV (R4) (R4) of FMDV: deletion of amino acids 84-143 of non-structural protein 3A, substitution of 3B1 and 3B2 with a codon-optimized 3B3 encoding sequence, resulting in deletion of 3B1/3B2 and duplication of 3B3, thereby introducing 3A&3B bimolecular labeling; meanwhile, enzyme cutting sites Pst I are introduced at two sides of a structural protein coding region and used for replacing coding genes of structural protein P1 of any other FMDV strain, and mutations for determining virus capsid stability are introduced in the structural protein coding region, so that 3A is obtained&3B-tagged full-length cDNA infectious cloning plasmid of capsid-stabilized FMDV avirulent strains, named pIRES_Bd43AB_mPst I, the recombinant virus rescued by this plasmid was designated rIRES_Bd43AB_m-Panasia. On the infectious cDNA cloning platform, O-type southeast Asia genotype Mya-98 strain O/M98/CHA/2010 and A-type Sea-97 genotype G2 subtype strain A/JLYS/CHA/2014 are selected according to the current prevalence situation of foot-and-mouth disease in Asia, and the P1 genes modified by heat resistance are respectively used for replacing pIRES_Bd43AB_mP1 gene of Pst I, and the constructed recombinant plasmids were designated pIRES_Bd43AB_m-O/M98 and pIRES_Bd43AB_m-A/J14. After the recombinant plasmids are verified to be correct by DNA sequence determination, the genome RNA of the recombinant virus is obtained by in vitro transcription, and BHK-21 cells are transfected to save the recombinant virus which is respectively named as rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_m-A/J14. Further, the 3 recombinant viruses are continuously transmitted for 10 generations on BHK-21 cells, and the nucleotide sequence determination and analysis prove that the recombinant viruses have high genetic stability. Importantly, the introduced 3A&The 3B molecular marker, the substituted IRES domain 4, and the thermotolerant phenotype modification site can all be stably inherited.

The constructed recombinant marker virus is compared with the in vitro growth characteristics of a wild-type virus O/YS/CHA/05 and a chimeric virus FMDV (R4) to find that the recombinant marker virus contains 3A&3B bimolecularly labeled recombinant virus rIRES_Bd43AB_m-PanAsia、rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mThe replication kinetics of-A/J14 on BHK-21 cells were similar, however, the peak replication time of these recombinant marker viruses was delayed by about 4 hours compared to wild-type viruses. For labeling 3A of virus&The 3B bimolecular marker was used for functional verification, and 3A was labeled with the 3A-specific monoclonal antibody 3A10 recognizing the marker molecule and the 3B-specific monoclonal antibody 2H1 previously prepared by the present inventors&3B labeled virus infected cells for indirect immunofluorescence detection. The immunofluorescence detection result shows that in the virus-infected BHK-21 cells, 3A&The 3B marker virus was recognized by FMDV shared mAb 10B10(Yang et al, Archives Virology, 2017; CN107177558A) as was the wild-type strain O/YS/CHA/05; however, mAb 2H1 recognizing the 3B protein and mAb 3A10 recognizing the 3A protein recognized only the wild-type virus O/YS/CHA/05, but not 3A&3B negatively tagged recombinant viral rIRES_Bd43AB_m-PanAsia、rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mA/J14, which functionally indicates the 3A of the recombinant marker virus of the invention&The 3B molecule negative label has been successfully introduced; meanwhile, Western blot analysis of virus infected cells by the monoclonal antibodies also proves the immunofluorescence detection result, which shows that 3A&FMDV negatively labeled with the 3B epitope lost reactivity to mab 3a10 and 2H 1.

The invention further uses a heat inactivation test to determine the heat stability phenotype of the recombinant marker virus prepared by the invention, and the determination result shows that the heat-resistant modified recombinant marker virus has obvious capsid heat-resistant stability characteristics.

To analyze the virulence of susceptible animals against recombinant marker viruses carrying a thermotolerant phenotype, the present invention uses higher doses of WT disease causing virus (10)⁵TCID₅₀Perhead) 10000 times rIRES_Bd43AB_mO/Panasia Virus dose (10)⁹TCID₅₀One) pigs are inoculated in the hoof skin for pathogenicity evaluation; the results show that the recombinant marker virus rIRES_Bd43AB_mthe-O/PanAsia did not replicate in pigs and could not establish infection, thus demonstrating that the recombinant marker virus carrying the thermotolerant phenotype was not pathogenic to pigs.

For detection of IRES Domain 4 replacement carrying a thermotolerant phenotype and 3A&3B negatively-tagged viral rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mThe virulence of the A/J14 is evaluated on three susceptible animals of pigs, cattle and sheep by adopting high dose and sensitive ways; the results indicate that IRES domain 4 was replaced carrying the thermotolerant phenotype and 3A&3B-tagged viral rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mthe-A/J14 strain is a non-virulent strain of FMDV because it has no replication ability in pig, cattle and sheep and thus completely loses pathogenicity. These results also show that the universal infectious cloning plasmid platform pIRES constructed using the present invention_Bd43AB_m-Pst I can be used for rapidly creating the dominant epidemic strain of FMDV carrying a heat-resistant phenotype and 3A in one country or region&3B negative-labeled avirulent strains used as seed viruses for inactivated vaccine production.

To evaluate the 3A carrying thermotolerant phenotype&3B-labeled FMDV avirulent strains, the invention relates to recombinant marker virus rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mThe A/J14 is inactivated by BEI and then mixed with ISA 201VG adjuvant in equal volume to prepare an O/A bivalent inactivated vaccine, and a commercial O/A bivalent inactivated vaccine (Zhongnongweite) is used as a vaccine control to be respectively inoculated to 10 pigs, and the other 6 pigs are inoculated with PBS as a negative control; the results of the immunoprotection efficacy test show that all inoculations of the inventionAnimals with bivalent inactivated vaccine detected neutralizing antibodies specific to type O and type A FMDV 7 days after vaccination (dpv), and the level of neutralizing antibodies reached a peak 21 days after vaccination (1: 256). The bivalent inactivated vaccine of the invention and the commercial bivalent inactivated vaccine induce the pig to generate the FMDV neutralizing antibody at a level similar to that of the FMDV neutralizing antibody, but the animals inoculated with PBS do not detect the FMDV neutralizing antibody, and no side reaction occurs after 26 pigs are inoculated. At 28 days post-immunization, the immunized animals were divided into two groups, challenged with homologous wild-type FMDV strains, type O and type a, respectively, and the effect of immune protection was observed. PBS-inoculated 6 control pigs were divided into 2 groups of 3 pigs each, inoculated with 10^8.5TCID₅₀Typical clinical symptoms of FMD occurred 1-3 days after either O-type FMDV O/M98/CHA/2010 or A-type FMDV A/JLYS/CHA/2014 per head dose. However, the bivalent inactivated vaccine prepared by the FMDV non-virulent strain has no FMD clinical manifestation and obtains immune protection after the inoculated animals are attacked by the O-type FMDV or the A-type FMDV, as the commercialized bivalent inactivated vaccine. Thus, the invention was created to carry the thermotolerant phenotype and 3A&3B-labeled FMDV non-virulent Strain rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mthe-A/J14 has excellent immunogenicity, can induce and generate high-level neutralizing antibodies when being inoculated to animals, can effectively resist the attack of parental virulent strains, and can be used as seed viruses for producing foot-and-mouth disease inactivated vaccines.

In conclusion, the invention constructs and saves the infectious cDNA clone plasmid pIRES of the safe, stable and negative-labeled foot-and-mouth disease inactivated vaccine seed virus_Bd43AB_m-Pst I carrying a molecular determinant of the thermotolerant phenotype of the viral capsid, a molecular determinant of the replication incompetent phenotype of the virus in vivo and the viral nonstructural protein 3A&3B epitope deletion negative marker elements, and Pst I enzyme cutting sites are introduced on both sides of the coding region of the virus structural protein P1 for rapid cloning and replacement of the structural protein coding region of any other foot-and-mouth disease virus strain. Recombinant virus rIRES constructed by replacing capsid protein coding regions of two current dominant epidemic strains by using the universal plasmid_Bd43AB_mO/M98 and rIRES_Bd43AB_m-a/J14, having the following characteristics: normal replication on BHK-21 cells; modified by heat resistanceThe stability of the post-viral capsid is significantly enhanced; with high doses (10)⁹TCID₅₀First) the virus does not infect pigs, cattle and sheep when being inoculated by a sensitive way; the seed virus thus prepared has the following excellent characteristics: not only solves the safety hidden trouble of virus dispersion in factories in the production of the foot-and-mouth disease inactivated vaccine, but also greatly reduces the production cost because the production process of the vaccine does not need high-standard biological safety protection; the stability of the 146S capsid is greatly improved by carrying out heat resistance modification on the virus capsid, thereby being beneficial to maintaining the immunogenicity of the inactivated vaccine and storing the prepared finished vaccine, improving the quality of the vaccine and reducing the cost of the vaccine; the matched 3B monoclonal antibody blocking ELISA method is used for detecting the serum of the immunized animal, so that the differential diagnosis of the vaccinated animal and the naturally infected animal can be realized, and the requirements of group clearing and purification in the foot-and-mouth disease immunization control plan can be met.

The two recombinant viruses are used as seed viruses to prepare the O/A bivalent inactivated vaccine, and immune animals can induce high-level neutralizing antibodies and obtain immune protection by using homologous strain attack; the blocking ELISA method established by the 3A and 3B monoclonal antibodies is used for detecting the vaccinated animals, so that the differential diagnosis of the vaccinated animals and naturally infected animals can be realized. The safe, stable and negative mark-carrying FMDV virus-free strain preparation method provided by the invention can be used for quickly creating seed viruses of inactivated vaccines in 2 weeks aiming at any FMDV epidemic strain, is used for preparing safe, stable and differential diagnosis-capable FMDV inactivated vaccines, and can provide a safe, effective and practical technical means for prevention, control and purification of global foot-and-mouth disease.

Drawings

FIG. 1 immunofluorescence assay to examine the binding ability of FMDV non-structural protein 3B mutants to monoclonal antibody 2H 1.

FIG. 2 selection of thermostable FMDV mutants and determination of their molecular determinants.

FIG. 33A &3B schematic construction of marked FMDV avirulent strains.

Figure 4 recombinant FMDV strain 3A &3B markers and validation of thermotolerant phenotype and its in vitro growth characteristics.

FIG. 5 shows the results of O/A type foot-and-mouth disease bivalent inactivated vaccine prepared from 3A &3B labeled heat-resistant recombinant FMDV non-virulent strain, 3B antibody response of commercial inactivated bivalent vaccine inoculated animal and monoclonal antibody blocking ELISA differential diagnosis.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Test materials

Cells, strains and antibodies: BHK-21 cells in 5% CO₂The culture was carried out at 37 ℃ in DMEM containing 10% FBS. The O-type FMDV O/YS/CHA/05 strain (GenBank accession number: HM008917) and the infectious cDNA clone pYS thereof are disclosed in the Chinese patent application No. CN101838658A (patent application No. 201010160669.9). FMDV (R4) infectious cDNA clone pFMDV (R4) is described in Chinese invention patent (CN108085302A (application No. 201711093774.3)) and International PCT patent (International publication No. WO/2018/090994A 1). A/QSA/CHA/2009, its nucleotide sequence homology with VP1 gene of reference strain A/HuBWH/CHA/2009(GenBank accession: JF792355) is up to 97.6%, its full-length cDNA infectious clone is pQSA. FMDV strain O/M98/CHA/2010, has up to 99.2% nucleotide sequence homology with VP1 of reference strain O/BY/CHA/2010(GenBank accession number: JN 998085). A/JLYS/CHA/2014(Liang et al, Archives Virology,2016), which has up to 99.6% homology to the VP1 nucleotide sequence of reference strain A/GDMM/CHA/2013(GenBank accession KF 450794). Monoclonal antibodies 10B10(Yang et al, Archives Virology, 2017; CN107177558A (application No.: 201710349810.1)), 3A10(Wang et al, Research in Veterinary Science, 2019; CN109295006A (patent application No.: 201811126938.2)) and 2H1(CN109295995A (application No.: 201811126187.4)) were prepared by the present inventors' laboratories.

EXAMPLE 1 evaluation of the pathogenicity of pigs with the FMDV (R4) Strain

Preliminary studies found that replacement of FMDV IRES domain 4(IRES-Fd4) with IRES chimeric virus FMDV (R4) generated by Bovine Rhinovirus (BRBV) IRES domain 4(IRES-Bd4) at 10⁶TCID₅₀The neck muscle inoculation of the natural host pig with the dose does not show any clinical symptoms of foot and mouth disease, does not produce viremia, does not expel toxin from oral cavity and nasal cavity, and does not induce antibodies in the pig body, which indicates that the virus loses replication capacity in susceptible animals, and the details are shown in Chinese invention patent (CN108085302A) and international PCT patent (application publication number: WO/2018/090994). To further evaluate the virulence of FMDV (R4) in pigs, the most sensitive heel intradermal route was used, with a higher FMDV (R4) vaccination dose (10)⁷TCID₅₀Head or 10⁸TCID₅₀Head) and inoculated in the same way 10⁵TCID₅₀Wild-type virus O/YS/CHA/05 at a head dose served as a virulent control, with the results shown in Table 1. Inoculation 10⁵TCID₅₀Pigs (11#, 12# and 13#) with wild type viruses have typical foot and mouth disease symptoms 2 days after inoculation, the body temperature is higher than 40 ℃, appetite is reduced, spirit is depressed, and blisters appear on four feet and the nose; the copy number of the virus RNA in blood, mouth and nose swabs 1 day after virus inoculation is obviously higher than the background value (2.6 log) of healthy pigs by using a Real-time PCR method to detect the virus RNA₁₀viral RNA CN/ml) which shows that viremia is formed and is discharged through oral cavity and nasal cavity when the pigs are inoculated, and the viremia and the discharge of the oral cavity and the nasal cavity reach the peak 3 to 4 days after inoculation; 10 days after inoculation, the inoculated pigs all produced high levels of neutralizing antibodies (>1:128). However, via the same heel intradermal route, 100-fold higher than wild-type virus titers (10)⁷TCID₅₀FMDV (R4) dose of/head) vaccinated pigs (01#, 02#, 03#, 04#, 5#), even for a 1000-fold (10 #) higher than wild-type virus titer⁸TCID₅₀(R4) dose of FMDV (R4) inoculated pigs (06#, 07#, 08#, 09#, 10#), no body temperature rise (all below 40 ℃) and no FMD clinical manifestation occurred in 10-day observation period, no virus RNA was detected in blood and oral-nasal swab samples collected every day by Real-time PCR, and no virus neutralizing antibody was generated in serum: (R4)<1:8). Inoculation test of this ultra-high dose, most sensitive routeThe experiment further showed that FMDV (R4) does not replicate in pigs, is unable to establish infection, and thus is completely debilitating to pigs.

TABLE 1 clinical presentation of pigs vaccinated with wild-type O/YS/CHA/05 and IRES chimeric virus FMDV (R4)

Example 23 comparison of the patterns of molecular markers on the B protein

In order to construct a molecular marker virus that can be used for differential diagnosis, this assay compared the pattern of molecular markers on the 3B protein. The inventor obtains a monoclonal antibody 2H1 aiming at FMDV non-structural protein 3B2 in earlier research in laboratories, and researches and determines the epitope recognized by the monoclonal antibody, wherein the epitope motif is³⁴KPLKVK³⁹，K³⁴、K³⁷And V³⁸Is a key amino acid of the epitope (CN109295005A, invention patent application No. 201811126187.4). In order to inactivate the epitope recognized by the monoclonal antibody 2H1 and introduce an antigen molecular marker, the key amino acid K of the 2H1 epitope is simultaneously replaced by alanine (A)³⁷And V³⁸Constructing the eukaryotic expression plasmid pCI-3B1 with inactivated 3B1 epitope_m23. In addition, analysis of the antigenicity of the 3B protein using DNAstar software showed that its epitopes were mainly concentrated in the 3B1 and 3B2 portions and that the 3B3 portion contained fewer epitopes. Because 3B1 and 3B2 are important for FMDV replication and will seriously affect FMDV replication after deletion, another 3B3 protein is selected to replace 3B1 and 3B2 proteins; in order to maintain the genetic stability of the virus and prevent the homologous recombination of two gene sequences encoding 3B3, the 3B3 protein coding sequence added instead of 3B1 and 3B2 was subjected to gene optimization (the optimized gene sequence is shown in SEQ ID NO. 1). The optimized 3B3 encoding gene is introduced into a recombinant virus produced by infectious clone pYS of O-type FMDV, and the recombinant virus is replicatedThe capability is similar to that of the parent strain; this modified 3B coding sequence was then used to construct a eukaryotic expression vector, designated pCI-3B 33. The eukaryotic expression plasmid pCI-3B1 for expressing the 3B protein mutant_m23 and pCI-3B33 and a control plasmid pCI-3B123(WT) expressing wild-type 3B protein were transfected into BHK-21 cells, 48H later, indirect immunofluorescence was detected using 2H1 antibody. The indirect immunofluorescence detection result is shown in figure 1, the wild type 3B123 protein can be combined with the 2H1 antibody and generates a strong specific fluorescence signal, and the mutant 3B1_mThe 23 protein can be weakly bound with the 2H1 antibody to generate a weak fluorescent signal, while the 3B33 protein is not completely bound with the 2H1 antibody and does not show any fluorescent signal. The test results show that the method and the strategy are feasible for replacing the 3B1 and 3B2 protein with the 3B3 protein which is optimized by the gene to remove the main antigenic region of the 3B protein so as to realize the molecular negative marker of the foot-and-mouth disease virus and the differential diagnosis of the vaccinated animal and the naturally infected animal by using the antibody detection ELISA established by the monoclonal antibody 2H 1.

Example 3 screening of genetically Stable FMDV Heat-resistant mutants and determination of molecular determinants of Heat-resistant phenotype

Test method 1

1.1 method for screening foot-and-mouth disease viruses under thermal stress

The hot pressure screening of viruses was carried out by heating at a specific temperature for 30 minutes to inactivate the virus titer of 99.99% per unit volume, and repeating the heat inactivation test at gradually increasing temperatures until the virus titers before and after heating were approximately the same. Multiple rounds of heat screening were performed at 51 deg.C, 53 deg.C and 56 deg.C until the emergence of highly heat-adapted mutant virus strains.

1.2 Virus capsid stability detection method

The virus is processed at higher temperature, and then the survival rate of the 146S complete virus capsid is determined, which is a key index for judging the thermal stability of the 146S virus antigen. Diluting the packaged hot-pressure-adapted virus and parent strain virus to 10 deg.C⁷TCID₅₀The preparation method comprises performing heat inactivation treatment at 42 deg.C, sampling at 15min, 30min, 45min, 60min, 120 min, 180 min, and 240min, and immediately standingCooling in ice bath, measuring the content of the virus 146S by adopting a sucrose density gradient centrifugation method, and calculating the retention ratio of the complete virus 146S capsid after heat inactivation in the ice bath.

2. Test results

Abhay Kotech et al found that the S2093Y mutation significantly increased the thermostability of FMDV capsids (Abhay Kotech et al, nature structural & molecular biology 2015). Subsequently, Katherine A.Scott et al found that the S2093Y mutation was not stably inherited as a molecular determinant of the heat-resistant phenotype of SAT 2FMDV (Katherine A.Scott et al, Journal of Virology, 2016). In view of the above, the test uses the full-length cDNA infectious clone plasmids pYS and pQSA of O-type and A-type FMDV to respectively construct and save heat-resistant mutants rO/YS-S2093Y and rA/QSA-Q2093C of the VP2 protein with amino acid site-directed mutation at position 93, and finds that the heat-resistant phenotypes are also unstable, the heat-resistant performance of the virus is gradually weakened in the process of in vitro passage, and the heat-resistant performance is completely disappeared when the virus is transmitted to the 5 th generation; sequencing of passage viruses of different generations shows that the mutant rO/YS-S2093Y has S2093Y mutated to S2093H when the mutant rA/QSA-Q2093C has the reversion mutation of Q2093C when the mutant rA/QSA-Q2093C is transmitted to the 5 th generation, thereby causing the heat-resistant phenotype of the mutant to disappear. The mutation studies of O-type and A-type FMDV of the present invention support the research results of Katherine A et al in SAT 2-type FMDV, further indicating that amino acid 93 of VP2 protein found by Abhay Kotecha et al as a determinant for thermotolerant phenotype molecules is not stably inherited.

In order to obtain genetically stable FMDV heat-resistant mutants, the test performs multiple rounds of hot-pressure screening on mutant viruses rO/YS-S2093Y and rA/QSA-Q2093C, and finally obtains O type FMDV heat-resistant mutants rO/YS delta and A type FMDV heat-resistant mutants rA/QSA delta (FIGS. 2A and 2B). Heat-resistant mutant strains rO/YS delta and rA/QSA delta obtained by heat-resistant pressure screening are respectively subjected to heat stability detection (action at 56 ℃ for 1h) in BHK-21 cells for 10 generations without pressure, and the heat resistance of the 10 th (P10) virus and the primary (P0) virus of the two heat-resistant mutant strains is not changed (figure 2), so that the heat-resistant phenotypes of the two heat-resistant mutant strains have genetic stability. Sequencing the structural protein coding region of the heat-resistant mutant showed that: the P10 generation virus of the O type FMDV heat-resistant mutant rO/YS delta only contains Y2079H and S2093Y 2 amino acid mutations like the P0 virus; like the P0 virus, the P10 generation virus of the heat-resistant mutant rA/QSA delta contains only 2 amino acid mutations of A1003T (the 3 rd amino acid A of VP1 protein is mutated into T) and N1017H (the 17 th amino acid N of VP1 protein is mutated into H), while Q2093C of the mutant virus has a back mutation. Cloning pYS and pQSA with O-type and A-type FMDV infectious cDNA, respectively, and performing single mutation and combined mutation on mutation sites of the heat-resistant mutant strains to determine molecular determinants for heat resistance of O-type and A-type FMDV. As a result, as shown in fig. 2C and 2D, Y2079H mutation alone did not increase the thermotolerance of type O FMDV (fig. 2C), and no thermotolerance analysis was performed because S2093Y mutation alone was not stably inherited; however, the Y2079H and S2093Y combined mutations significantly enhanced the heat tolerance properties of type O FMDV (fig. 2C). The a1003T and N1017H mutations alone did not increase the thermotolerant characteristics of type O FMDV, while the combination mutations significantly enhanced the thermotolerant characteristics of type a FMDV. The above experimental results show that the Y2079H mutation, although not producing a thermotolerant phenotype, can be stably inherited in synergy with the S2093Y thermotolerant mutation, thus synergistically determining the capsid thermotolerant stability phenotype of O-type FMDV; the a1003T and N1017H mutations synergistically determine the capsid thermostable phenotype of type a FMDV and are stably heritable.

Example 4 construction and rescue of FMDV avirulent strains carrying thermostable phenotype and negative marker for deletion of non-structural protein 3A &3B epitopes, verification of thermostable stability and detection of in vitro growth characteristics thereof

Test method 1

1.13 construction of full-Length cDNA infectious clones of FMDV-null strains labelled with A &3B

3A&Construction of full-length cDNA infectious clones of 3B bimolecular-labeled FMDV avirulent strains, the procedure was as follows: firstly, using pYS as a template, carrying out PCR amplification by using 5458(EcoR I) -F and d3AB-1-R primers, carrying out gel recovery and purification on the obtained PCR product, using the primers 5458(EcoR I) -F, d3AB-2-R and d3AB-3-R as templates for amplification, and obtaining a PCR product called fragment A; then using pYS as a template, amplifying by using primers d3B-2H1-F and 8221(EcoR V) -R, and obtaining a PCR product called fragment B; finally, the purified A, B fragment was used as a template, and primers 5458(EcoR I) -F and 8221(EcoR V) -R were usedFusion PCR amplification to produce a fragment of approximately 2.4Kb in size lacking amino acids 84-143 of the 3A protein and replacing 3B1 and 3B2 with a codon-modified 3B 3; the 2.4Kb fragment was double digested with EcoR I and EcoR V, cloned into the same digested pFMDV (R4) vector, and the clone determined to be correct by sequence analysis was named pIRES_Bd43AB_m(ii) a Then, the pIRES is used_Bd43AB_mAs a template, primers L (pst I) -F and L (pst I) -R, 2A (pst I) -F and 2A (pst I) -R were used for PCR amplification, PstI cleavage sites were introduced on both sides of the coding region of the structural protein P1, and a clone determined to be correct by sequence analysis was named pIRES_Bd43AB_m-Pst I。

Constructing genome cDNA of FMDV strains O/M98/CHA/2010 and A/JLYS/CHA/2014, respectively serving as templates, respectively using P1-F and O/M98P1-R or A/J14P1-R as primers, respectively amplifying P1 genes of the 2 strains and carrying out heat resistance mutation on the genes, respectively cloning the products to pIRES (plasmid reduction) digested by Pst I by using In-fusion recombinase (Clontech) after PCR amplification and gel recovery and purification of the products_Bd43AB_mIn the-Pst I vector, the recombinants were named pIRES respectively after being confirmed to be correct by sequence analysis_Bd43AB_m-O/M98 and pIRES_Bd43AB_m-A/J14. The primers used in the present invention are shown in Table 2.

TABLE 2 primers and sequences thereof for constructing 3A & 3B-labeled FMDV avirulent strains

1.2 rescue of Virus

Recombinant plasmid pIRES_Bd43AB_m-O/M98 and pIRES_Bd43AB_mthe-A/J14 was linearized by the restriction enzyme EcoRV, according to RiboMAX^TMThe in vitro transcription was performed by the Large Scale RNA Production Systems-T7 system instructions, in the following reaction system: 25mmol/L rNTP 6 mu L,5 Xbuffer 4 u L, T7RNA polymerase mixture 2L, linear recombinant plasmid 8L (2 u g), the total volume is 20L. The reaction was mixed well, incubated at 37 ℃ for 2.5h, digested with RNase-Free DNase for 15min, the DNA template removed, and the in vitro transcription product purified by phenol chloroform extraction. When BHK-21 cells in 6-well plates grew to 80% -90% monolayer, cells were washed 2 times with PBS and 1.5mL of DMEM cell culture containing 2% fetal bovine serum was added. BHK-21 cells were transfected with RNA obtained by in vitro transcription according to the instructions of the Effect Transfection Reagent kit from QIAGEN for virus rescue. Transfected cells at 5% CO₂Culturing at 37 deg.C, observing cytopathic effect, harvesting virus about 3 days, repeatedly freezing and thawing for 3 times, inoculating BHK-21 cell, and passaging until virus can produce stable CPE. Obtaining rescued recombinant virus, verifying the strain with correct sequence by full-length genome sequence determination, and respectively naming the strain as rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mA/J14, for subsequent experiments.

1.3 plotting Virus growth curves

Wild type virus O/YS/CHA/05, FMDV (R4) and recombinant virus rIRES_Bd43AB_m-PanAsia,rIRES_Bd43AB_m-O/M98、rIRES_Bd43AB_mA/J14, inoculating BHK-21 cells in log phase at 0.05MOI dose, adsorbing at 37 deg.C for 1h, washing off unadsorbed virus with PBS, adding DMEM containing 2% fetal calf serum to maintain culture, harvesting culture supernatants at 2h, 4h, 6h, 8h, 10h, 12h, 16h and 20h after inoculation, and determining TCID of harvested virus at different time points₅₀Titers, 3 replicates per time point, and mean values were calculated. The time of virus infection of cells is used as the abscissa, and the TCID of the virus at different time points₅₀The log of titers is the ordinate and the growth curve of virus replication is plotted.

1.4 in vitro passage and genetic stability detection of recombinant FMDV Virus strains

The recombinant FMDV strains were inoculated to BHK-21 cells at a dose of 0.05MOI, adsorbed at 37 ℃ for 1 hour, washed 2 times with PBS, and maintained in culture with DMEM containing 2% fetal bovine serum. After obvious cytopathic effect appears in the cells, the cells are repeatedly frozen and thawed for 3 times, and the supernatant is obtained by centrifugation and is passaged to the 10 th generation in the BHK-21 cells. Extracting virus RNA, performing RT-PCR amplification and sequence determination.

1.5Western blot detection

After the BHK-21 cells inoculated with the virus are cultured for 12h, the cells are harvested and subjected to lysis treatment, and the cells are transferred to a nitrocellulose membrane after SDS-PAGE electrophoresis. Blocking the membrane with 5% skim milk, incubating with MAb 10B10, 3A10 or 2H1 (1: 1000 dilution) as primary antibody, respectively, performing incubation at 37 deg.C for 1H, washing with PBST, adding HRP-labeled anti-mouse IgG-antibody (anti-mouse IgG-HRP, 1: 5000 dilution) for 1H at 37 deg.C, and performing color development identification with ECL luminescence solution. The internal reference was made to the beta-actin antibody (1: 1000 dilution) as the primary antibody, and the anti-mouse IgG-HRP (1: 5000 dilution) was similarly used as the enzyme-labeled antibody.

1.6 Indirect immunofluorescence

BHK-21 cells grown in 96-well plates were inoculated with virus, rinsed 2 times with PBS after 6H, fixed with-20 ℃ absolute ethanol for 15min, added with monoclonal antibodies 10B10, 3A10 or 2H1 diluted 1:100, incubated in 37 ℃ wet box for 60min, washed 5 times with PBS, added with FITC-labeled goat anti-mouse IgG (1:100) and incubated in 37 ℃ wet box for 45min, mounted with 70% glycerol and visualized under a fluorescent microscope.

2. Test results

2.1 construction and rescue of FMDV Strain carrying thermostable phenotype and non-structural protein 3B negative marker

In order to construct an avirulent strain of foot-and-mouth disease virus with a differential diagnosis marker as a foot-and-mouth disease inactivated vaccine seed virus, the invention takes infectious cDNA clone plasmid pFMDV (R4) (figures 3A and 3B) of FMDV (R4) as the basis to carry out the following genetic engineering operations:

deletion of amino acids 84-143 of non-structural protein 3A and substitution of 3B1 and 3B2 with a codon optimized 3B3 coding sequence, resulting in deletion of 3B1/3B2 and duplication of 3B3, thereby introducing a molecular marker (fig. 3C, 3D); meanwhile, enzyme cutting sites Pst I are introduced on two sides of the structural protein coding region, so that the coding gene of the structural protein P1 of any other FMDV strain can be replaced, and the structural protein coding region is introduced to determine the stability of the virus capsidThereby obtaining 3A&3B-tagged FMDV non-virulent strain full-length infectious cDNA clone plasmid named pIRES_Bd43AB_mPst I, the recombinant virus rescued by this plasmid was designated rIRES_Bd43AB_m-PanAsia。

On the infectious cDNA cloning platform, O-type southeast Asia genotype Mya-98 strain O/M98/CHA/2010 and A-type Sea-97 genotype G2 subtype strain A/JLYS/CHA/2014 are selected according to the current prevalence situation of foot-and-mouth disease in Asia, and the P1 genes modified by heat resistance are respectively used for replacing pIRES_Bd43AB_mP1 gene of Pst I, and the constructed recombinant plasmids were designated pIRES_Bd43AB_m-O/M98 and pIRES_Bd43AB_m-A/J14. After the recombinant plasmids are verified to be correct by DNA sequencing, the genome RNA of the recombinant virus is obtained by in vitro transcription, and the recombinant virus is rescued by transfecting BHK-21 cells and is respectively named as rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mA/J14 (FIG. 3A). Furthermore, the 3 recombinant viruses were continuously transmitted for 10 generations on BHK-21 cells, and nucleotide sequence determination and analysis confirmed that the recombinant viruses had high genetic stability. Importantly, the introduced 3A&The 3B molecular marker, the replaced IRES domain 4, and the thermostable modification site can all be stably inherited.

2.2 3A &3B labelling of recombinant FMDV and verification of thermostability and in vitro growth characteristics

Contains 3A compared with the in vitro growth characteristics of wild-type virus O/YS/CHA/05 and chimeric virus FMDV (R4)&3B bimolecularly labeled recombinant virus rIRES_Bd43AB_m-PanAsia、rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mThe replication kinetics of-A/J14 on BHK-21 cells were similar, however, the peak time of replication of these marker viruses was delayed by about 4 hours compared to wild-type viruses (FIG. 4A).

To treat 3A of recombinant virus&3B bimolecular labeling was performed for functional verification using the specific monoclonal antibody 3A10 (Chinese patent publication No. CN109295006A (patent application No. 201811126938.2)) recognizing the labeled molecule 3A prepared by the present inventors and the specificity recognizing the 3BMonoclonal antibody 2H1 (Chinese patent CN109295005A (patent application No.: 201811126187.4)), p-3A&3B labeled virus infected cells for indirect immunofluorescence detection. The epitope recognized by 3A10 is known to be the 5-aa peptide sequence located on the FMDV 3A protein¹²⁶ERTLP¹³⁰(Wang et al, Research in Veterinary Science, 2019; Chinese patent application publication No. CN109293748A (patent application No.: 201811126944.8)), so monoclonal antibodies 3A10 and¹²⁶ERTLP¹³⁰the missing 3A protein loses reactivity; while the epitope recognized by 2H1 is the 6-aa peptide sequence located on FMDV 3B2 protein³⁴KPLKVK³⁹(patent application publication No. CN109293747A (patent application No. 201811126936.3)), so mAb 2H1 and³⁴KPLKVK³⁹the missing 3B2 protein lost reactivity. The results of immunofluorescence assay are shown in FIG. 4B, 3A in virus-infected BHK-21 cells&The 3B marker virus, like the wild-type strain O/YS/CHA/05, was recognized by FMDV shared monoclonal antibody 10B10(Yang et al, Archives Virology, 2017; Chinese patent application publication No. CN107177558A (patent application No. 201710349810.1)); however, mAb 2H1 recognizing the 3B protein and mAb 3A10 recognizing the 3A protein recognized only the wild-type virus O/YS/CHA/05, but not 3A&3B negatively tagged viral rIRES_Bd43AB_m-PanAsia、rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mA/J14 (FIG. 4B), which functionally indicates 3A of the recombinant virus&3B molecular markers have been successfully introduced. At the same time, Western blot analysis of virus-infected cells with these monoclonal antibodies (FIG. 4C) also confirmed the immunofluorescence assay results, indicating 3A&FMDV negatively labeled with 3B lost reactivity to mab 3a10 and 2H 1.

The thermal stability of the labeled virus was determined using a heat inactivation assay. The titer is about 10⁷TCID₅₀The cell culture supernatants of the wild-type virus and the labeled virus, in a/ml format, were heat-inactivated at 42 ℃ for 4h, and the proportion of intact 146S FMDV particles in the heat-treated samples was quantified by FMDV146S sucrose density gradient centrifugation. The results are shown in FIGS. 4D, 4E and 4F, for the marker virus rIRES_Bd43AB_m-A/J14、rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mThe percentages of intact 146S particles retained by PanAsia after 4h of treatment at 42 ℃ were 68.9%, 85.7% and 82.2%, respectively, while the percentages of 146S particles retained by their parental wild-type viruses were 3.6%, 5.3% and 5.6%, respectively, and the results indicated that the thermotolerant modified recombinant marker viruses had significant capsid thermotolerant stability characteristics.

EXAMPLE 5 evaluation of virulence of recombinant Virus strains in susceptible hosts

1. Test method

1.1 pig toxicity evaluation test

Healthy piglets 34, 20-30 kg, and negative serum FMDV antibody. Randomly selecting 9 heads to be divided into 3 groups, wherein each group comprises 3 heads: first, the heel of a pig's hoof was inoculated intradermally with the wild type strain FMDV O/YS/CHA/05 at a dose of 10⁵TCID₅₀A/head; second and third groups, heel of pig hoofs were inoculated intradermally with wild-type strains O/M98/CHA/2010 or A/JLYS/CHA/2014 at a dose of 10⁷TCID₅₀Head of the device. The remaining 25 pigs were randomly divided into 5 groups of 5 pigs each, and 10 pigs were inoculated into the heel skin⁷TCID₅₀Head or 10⁸TCID₅₀FMDV for one dose (R4), 10⁹TCID₅₀First dose of recombinant virus rIRES_Bd43AB_m-Panasia or rIRES_Bd43AB_m-O/M98 or rIRES_Bd43AB_m-A/J14. The body temperature of the vaccinated pigs was measured, clinical performance was observed and nasal swabs, oral swabs and blood were collected daily for 10 days after vaccination.

1.2 bovine toxicity evaluation test

Healthy Holstein cows 16, 200 and 250 kg, serum FMDV antibody negative. Randomly selecting 6 heads, dividing into two groups of 3 heads, and respectively inoculating 10 doses into the cow tongue skin⁷TCID₅₀Wild-type strain O/M98/CHA/2010 or A/JLYS/CHA/2014/cephalin. The rest 10 cattle are divided into two groups of 5 cattle, and the inoculation dose in the lingual skin of each group is 10⁹TCID₅₀First recombinant virus rIRES_Bd43AB_m-O/M98 or rIRES_Bd43AB_m-A/J14. The body temperature of the cattle was measured, clinical performance was observed, and nasal swab and oral swab were collected every day within 10 days after inoculationAnd blood.

1.3 sheep toxicity evaluation test

The healthy sheep are 16, 30-40 kg, and the FMDV antibody in the serum is negative. Randomly selecting 6 hoof, dividing into two groups of 3, injecting 10 intradermally into each group of hoof crown⁷TCID₅₀Wild type strain O/M98/CHA/2010 or A/JLYS/CHA/2014 at the head dose. The remaining 10 sheep were divided into two groups of 5, and the hoof crowns were injected intradermally with 10⁹TCID₅₀First dose of recombinant virus rIRES_Bd43AB_m-O/M98 or rIRES_Bd43AB_m-A/J14. Within 10 days after inoculation, the temperature of the sheep was measured, clinical performance was observed and nasal swabs, oral swabs and blood were collected daily.

2 results of the test

2.1 viral rIRES with replacement of IRES Domain 4, thermostable mutation, 3B labelling_Bd43AB_mThe (E) -O/Panasia has no pathogenicity to pigs

To analyze viral rIRES carrying a thermotolerant phenotype with IRES domain 4 replaced and a 3B marker_Bd43AB_mVirulence of-O/PanAsia in susceptible animals, 10000-fold higher titres (10) than the wild-type virus were used in this experiment⁹TCID₅₀Dose/head) pigs (15#, 16#, 17#, 18# and 19#) were inoculated into hoof skin for pathogenicity evaluation, and the results are shown in table 2. In the observation period, the temperature of the vaccinated pigs is not increased (the temperature is lower than 40 ℃), and the vaccinated pigs have no clinical FMD; detecting virus RNA in blood and mouth and nose swabs, wherein the virus RNA detected 1-10 days after inoculation is negative; in addition, no neutralizing antibodies to FMDV were produced in pigs during the observation period: (<1:8). This indicates that the marker virus rIRES_Bd43AB_mthe-O/PanAsia does not replicate in pigs, is unable to establish infection, and is therefore not pathogenic to pigs.

2.2 viral rIRES with replacement of IRES Domain 4, thermostable mutation, 3B labelling_Bd43AB_mO/M98 and rIRES_Bd43AB_mthe-A/J14 has no pathogenicity on pigs, cattle and sheep

For detection of viral rIRES carrying a thermotolerant phenotype with IRES Domain 4 replaced and a 3B marker_Bd43AB_mO/M98 and rIRES_Bd43AB_mVirulence of-A/J14, the virulence of these two recombinant viruses was evaluated in three susceptible animals, pig, cattle and sheep, respectively, and the results are shown in tables 3 and 4. First using wild type virus O/M98/CHA/2010 or A/JLYS/CHA/2014 at 10%⁷TCID₅₀The pigs (21#, 22# and 23#, or 31#, 32# and 33#) inoculated with the dose are used as a control, the body temperature rise and the typical foot-and-mouth disease symptoms occur 48h after inoculation, and the content of virus RNA in the blood and the mouth and nose swabs of the pigs 3-4 days after inoculation is obviously higher than that of healthy pigs (2.6 log)₁₀viral RNA CN/ml) indicating that severe viremia had occurred at this time, producing high loads of oral and nasal detoxification, inducing higher titers of neutralizing antibodies 10 days after vaccination. However, marker virus rIRES with IRES domain 4 replaced_Bd43AB_m-O/M98 or rIRES_Bd43AB_mA/J14, at 10⁹TCID₅₀The pigs (24#, 25#, 26#, 27#, 28#, or 34#, 35#, 36#, 37#, 38#) are inoculated with the highest dose through the most sensitive heel intradermal route, the clinical symptoms and the body temperature rise of FMD are not generated in the observation period, and the virus RNA is negative (the copy number is lower than 2.6 log) in the blood and mouth and nose swab samples collected 1-10 days after inoculation₁₀viral RNA CN/ml), no virus neutralizing antibody was produced during the observation period (<1:8). These results indicate that viral rIRES carrying the thermotolerant phenotype and a 3B marker with IRES domain 4 replaced_Bd43AB_mO/M98 and rIRES_Bd43AB_mthe-A/J14 is not replicating in pigs, is unable to establish an infection and is therefore completely non-pathogenic to pigs.

For detection of viral rIRES carrying a thermotolerant phenotype with IRES Domain 4 replaced and a 3B marker_Bd43AB_mO/M98 and rIRES_Bd43AB_mPathogenicity of A/J14 to cattle, first with wild-type virus O/Mya98/CHA/2010 or A/JLYS/CHA/2014 at 10⁷TCID₅₀The dose of the/head was administered orally to the tongue skin of cattle (01#, 02# and 03#, or 11#, 12# and 13#) as a positive control, which developed fever and viremia 3 days after inoculation, and typical foot and mouth disease clinical symptoms 4-6 days after inoculation (tables 3 and 4). However, diseases carrying a thermotolerant phenotype and a 3B marker in which IRES domain 4 is replacedToxic rIRES_Bd43AB_m-O/M98 or rIRES_Bd43AB_mA/J14 use 100-fold wild-type pathogenic Virus titre (10)⁹TCID₅₀Dose/head) was administered via the most sensitive lingual intradermal route to cattle (04#, 05#, 06#, 07 and 08 #; or 14#, 15#, 16#, 17# and 18#), no FMD clinical manifestations, body temperature elevation, no viremia, no oral and nasal toxicant expulsion (tables 3 and 4), and no virus neutralizing antibody production during the 10-day observation period (see table 3 and table 4) ((see table 1)<1:8). This indicates that viral rIRES carrying the thermotolerant phenotype and a 3B marker with IRES domain 4 replaced_Bd43AB_mO/M98 and rIRES_Bd43AB_mA/J14 does not replicate in cattle, is unable to establish infection, and thus is completely debilitating to cattle.

For detection of viral rIRES carrying a thermotolerant phenotype with IRES Domain 4 replaced and a 3B marker_Bd43AB_mO/M98 and rIRES_Bd43AB_mPathogenicity of A/J14 to sheep, wild-type virus O/Mya98/CHA/2010 or A/JLYS/CHA/2014 was first used at 10%⁷TCID₅₀The dose per head, sheep inoculated through the intradermal route of hoof and crown (01#, 02#, and 03#, or 10#, 11#, and 12#) as positive control, all sheep inoculated at 2-4dpi showed increased body temperature, hoof or tooth trace blisters, viremia, and oral and nasal detoxification (tables 3 and 4), but the onset of lameness was not obvious. However, viral rIRES carrying a thermotolerant phenotype and a 3B marker with IRES domain 4 replaced_Bd43AB_m-O/M98 or rIRES_Bd43AB_mA/J14 titers 100-fold for wild-type pathogenic virus via the most sensitive hoof crown intradermal route (10 ×)⁹TCID₅₀First) dose of vaccinating sheep (05#, 06#, 07#, 08# and 09 #; or 16#, 17#, 18#, 19# and 20#), and no FMD clinical manifestations, body temperature elevation, viremia, oral and nasal toxicant elimination (tables 3 and 4) and no virus neutralizing antibody production in a 10-day observation period (Table 3 and 4)<1:8). This indicates that viral rIRES carrying the thermotolerant phenotype and a 3B marker with IRES domain 4 replaced_Bd43AB_mO/M98 and rIRES_Bd43AB_m-A/J14, which does not replicate in sheep, is unable to establish infection, and therefore is completely non-pathogenic to sheep.

The above knotResults indicate that IRES Domain 4 is replaced with a viral rIRES carrying a thermotolerant phenotype and a 3B marker_Bd43AB_mO/M98 and rIRES_Bd43AB_mthe-A/J14 strain is said to be FMDV avirulent because it is replication-incompetent in pigs, cattle and sheep and thus completely pathogenic. These results also show that the universal infectious cloning plasmid platform pIRES of the present invention is used_Bd43AB_mPst I, which can rapidly create a virus-free strain carrying a non-toxic and heat-resistant phenotype and a 3B negative marker against the FMDV dominant circulating strain in a country or region, and is used as a seed virus for inactivated vaccine production.

TABLE 3 wild strain O/M98/CHA/2010 and modified Virus rIRES_Bd43AB_mClinical presentation of-O/M98 vaccinated porcine cattle and sheep

TABLE 4 wild strain A/JLYS/CHA/2014 and modified Virus rIRES_Bd43AB_mClinical presentation of-A/J14 vaccinated porcine cattle and sheep

Example 6 preparation of bivalent inactivated vaccine against heat-resistant and avirulent O/A type foot-and-mouth disease and immunoprotection potency test

Test method 1

1.1 preparation of bivalent inactivated vaccine against heat-resistant, avirulent O/A type foot-and-mouth disease

BHK-21 cells were cultured in suspension in a shake flask when grown to 3.5X 10⁶cells/ml, 0.01MOI inoculation of recombinant virus rIRES_Bd43AB_m-O/M98 or rIRES_Bd43AB_mSuspension culture at 37 deg.C for 16h at-A/J14 to obtain virus, freeze thawing for three times, and passing through TCID₅₀Drops for analytical determination of virusesAnd (4) degree. Diluting the virus to 10^9.25TCID₅₀After/ml BEI (final concentration 2mmol/L) was added and the inactivation was carried out at 30 ℃ for 28h, while shaking every 2h, after which the inactivation was stopped by adding a 2% sodium thiosulfate solution. After virus inactivation is qualified through inspection, rIRES is added_Bd43AB_mO/M98 and rIRES_Bd43AB_mAnd (3) mixing A/J14 in equal volume, adding Montanide ISA 201VG adjuvant (SEPPIC of France) in the same volume, mixing uniformly, preparing 3A/3B marked bivalent inactivated vaccine O/A of foot-and-mouth disease virus with capsid heat-resistant and stable by using an avirulent strain, and storing at 4 ℃ for later use.

1.2 immunization and challenge test in pigs

26 healthy mount pigs, 30-40 kg, and negative serum FMDV antibody. Randomly selecting 20 heads to be divided into 2 groups, wherein each group comprises 10 heads: one group was inoculated with O/A bivalent inactivated vaccine (rIRES) at the back of the ear and neck muscle (2 ml/head)_Bd43AB_mO/M98 Strain + rIRES_Bd43AB_m-a/J14 strain), another group of postauricular neck muscle inoculations (2 ml/head) with a commercial O/a bivalent inactivated vaccine (Re-O/MYA98/JSCZ/2013 strain + Re-a/WH/09 strain, zhongnongwitt), the remaining 6 pigs being inoculated with PBS as challenge control. At 28 days after inoculation, two vaccine experimental groups were randomly divided into 2 groups, each group selected 5 immunized pigs and 3 PBS inoculated control pigs, so that neck muscles of 8 pigs in each group were inoculated with 2ml 10 ml^8.5TCID₅₀O/M98/CHA/2010 strain or A/JLYS/CHA/2014 strain at a/ml dose. Within 10 days after challenge, body temperature was measured, clinical performance was observed and nasal swabs, oral swabs and blood were collected daily.

1.3 vaccine repeat immunization test

FMDV serum antibody-negative healthy pigs, cattle, sheep, 20 each, were randomly divided into 2 groups of 10. One group was inoculated with 2-fold the dose (4 ml/head) of the marked avirulent strain O/a bivalent inactivated vaccine, and the other group was inoculated with 2-fold the dose (4 ml/head) of the commercial O/a bivalent inactivated vaccine. Inoculating once every 28 days, continuously inoculating for 4 times, collecting serum of inoculated animal 30 days after last inoculation, and storing in refrigerator at-20 deg.C for use.

1.4 Observation of clinical symptoms

Animals were carefully observed daily for clinical morbidity and recorded, and clinical scores were made according to the method described by Elizabeth Rieder et al (Elizabeth Rieder et al, J Virol, 2005): hoof onset was scored 1 per hoof, nasal, lingual or lip onset was scored 1, and the maximum score was 5.

1.5 oral and nasal detoxification and detection of viremia

Collecting nasal swab, oral swab and serum sample, extracting total RNA by TRIZOL method, and extracting with Oligo (dT)₁₅) cDNA obtained by reverse transcription of the primers was used as a template, and FMDV-specific primers (3 DF: 5'GGA TGC CGT CTG GTT GTT 3'; 3 DR: 5'CGT AGG AGA TCA TGG TGT AAG AGT 3') were subjected to fluorescent quantitative PCR detection. Detailed operation of fluorescent quantitative PCR

Green qPCR Super Mix-UDG with ROX (Invitrogen) kit instructions, through the standard curve function to calculate the sample in the content of viral genomic RNA. FMDV RNA copy number 2.6 by PCR amplification from serum and mouth and nose swab samples of healthy pigs

Log10 was taken as background value, above which the number of copies of viral RNA/ml (viral RNA CN/ml) was judged as positive for FMDV RNA.

1.6 micro cell neutralization assay

The method of fixing virus diluted serum is adopted to carry out a trace cell neutralization test. First, TCID of FMDV was determined using BHK-21 cells₅₀(ii) a Then, inactivating the serum at 56 ℃ for 30min, and diluting by PBS; using 100TCID₅₀The virus is respectively mixed with the serum diluted by different dilutions with the same volume, and the mixture is incubated for 1h in an incubator at 37 ℃; the above incubated serum-virus mixtures were inoculated into BHK-21 cells at 100. mu.L per well, repeated at 8 wells per dilution, and incubated at 37 ℃ in 5% CO₂Culturing in an incubator, observing cytopathic effect (CPE) every day, and making final judgment after 72 h. In addition, virus, positive serum and normal cell control were set, and the virus neutralization titer was calculated according to the method of Reed-Muench (Reed and Muench.,1938) based on the CPE change, thereby protecting 50% BHK-21 cellsSerum dilution concentrations of CPE did not occur.

1.7 foot-and-mouth disease virus 3B monoclonal antibody blocking ELISA (CN109295005A, patent application No.: 201811126187.4)

The monoclonal antibody 3a10 was diluted with sodium carbonate buffer (pH 9.6) and coated on ELISA plates at 100 μ L/well overnight at 4 ℃; washing with PBST for 5 times, adding prokaryotic expression foot-and-mouth disease virus non-structural protein 3AB antigen, 50 μ L per well, and incubating at room temperature for 1 h; PBST is washed for 5 times, serum to be detected which is diluted by 10 times is added, 50 mu L/hole is formed, negative serum and positive serum are compared, incubation is carried out for 1h at 37 ℃, and PBST is washed for 5 times; adding HRP marked detection monoclonal antibody 2H1 with working concentration, 50 mu L/hole, and incubating for 1H at 37 ℃; PBST was washed 5 times, 50. mu.L of TMB substrate solution was added to each well and developed for 15min in the dark, an equivalent amount of concentrated sulfuric acid was added to terminate the reaction, and the light absorption value (OD) at 450nm wavelength was measured with a microplate reader_450nmValue). And (4) judging a result: calculating inhibition Percentage (PI) and judging whether the PI is negative or positive, wherein the PI is positive when the PI is more than or equal to 50 percent (negative control OD value-sample OD value)/(negative control OD value-positive control OD value), and the PI is positive<50% are negative.

2 results of the test

2.1 marked thermotolerant avirulent strain O/A bivalent inactivated vaccine of FMDV induces effective immunoprotection

To evaluate the 3A carrying thermotolerant phenotype&Immunoprotective efficacy of inactivated vaccines prepared from 3B-labeled FMDV avirulent strains, modified viral rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mBEI (Bei inactivation) of-A/J14, mixing with ISA 201VG adjuvant in equal volume to prepare O/A type foot-and-mouth disease bivalent inactivated vaccine, and using commercial O/A bivalent inactivated vaccine (Zhongnongweite) as vaccine control, respectively inoculating 10 pigs, and inoculating 6 pigs with PBS as negative control, wherein the results are shown in tables 5 and 6. Neutralizing antibodies specific to type O and type A FMDV were detected 7 days after vaccination (dpv) in all inactivated vaccinated animals, and the level of neutralizing antibodies reached a peak 21 days after vaccination (1: 256). The level of FMDV neutralizing antibodies induced by the O/A type foot-and-mouth disease bivalent inactivated vaccine inoculated pig (201# -210#) and the commercial bivalent inactivated vaccine inoculated pig (101# -110#) is similar, but the FMDV is not detected by the animal (301# -306#) inoculated with PBSAnd an antibody (a)<8). Moreover, no side reactions occurred in 26 pigs after vaccination.

At 28 days post-immunization, the immunized animals were divided into two groups, challenged with homologous wild-type FMDV strains, type O and type a, respectively, and the effect of immune protection was observed. PBS-inoculated 6 control pigs were divided into 2 groups of 3 pigs each, inoculated with 10^8.5TCID₅₀Typical clinical symptoms of FMD occurred 1-3 days after the first dose of either O-type FMDV O/M98/CHA/2010 or A-type FMDV A/JLYS/CHA/2014 (tables 3 and 4). However, the bivalent inactivated vaccine for type O/a foot and mouth disease prepared from the modified FMDV avirulent strain in this experiment, like the commercial bivalent inactivated vaccine, did not show clinical manifestation of FMD and acquired immune protection after challenge with type O or type a FMDV (table 5, table 6). Thus, the FMDV avirulent strain rIRES created by the present invention, which carries a thermotolerant phenotype and a 3B marker_Bd43AB_mO/M98 and rIRES_Bd43AB_mthe-A/J14 has excellent immunogenicity, can induce and generate high-level neutralizing antibodies when being inoculated to animals, can effectively resist the attack of parental virulent strains, and can be used as seed virus for producing foot-and-mouth disease inactivated vaccines.

TABLE 5 neutralizing antibodies and immunoprotection against O-type FMDV induced by vaccination of pigs with O/A bivalent inactivated vaccine

TABLE 6O/A bivalent inactivated vaccine inoculation pig induced neutralizing antibody of FMDV type A and immunoprotection

2.2 monoclonal antibody blocking ELISA detection of 3B antibody response in FMDV marker inactivated vaccinated animals

A small amount of non-structural protein can be remained in the FMD inactivated vaccine in the preparation process, and an animal can be induced to generate a certain level of non-structural protein specific antibody after repeated immunization, so that the differential diagnosis of the vaccinated animal and the naturally infected animal is interfered, and the vaccinated animal and the naturally infected animal cannot be subjected to the interferenceAnd performing effective identification. The invention uses the established FMDV 3B monoclonal antibody blocking ELISA antibody detection method, and completely eliminates the 3B epitope (short peptide positioned in 3B 2) recognized by the 2H1 monoclonal antibody through the deletion of the main antigen regions 3B1 and 3B2 of the FMDV virus-free strain 3B protein³⁴KPLKVK³⁹) Thus, the blocking ELISA method established by the invention can not detect the animals immunized by the inactivated virus vaccine, thereby eliminating the interference of residual 3B protein in the vaccine on differential diagnosis. In order to verify the differential diagnosis design of the marker vaccine, the test uses the FMDV O/A type bivalent inactivated vaccine with the 3B deletion negative marker to respectively inoculate three animals of a pig, a cattle and a sheep with 2 times dosage (4 ml/head), continuously immunizes the three animals for 4 times at intervals of 28 days, collects serum 30 days after the last immunization, evaluates the generation and the level of the non-structural protein 3B antibody by a blocking ELISA method established based on the 3B protein monoclonal antibody 2H1, simultaneously selects the serum of the animal inoculated with the commercial O/A bivalent inactivated vaccine (without the negative marker of the 3B deletion) as a detection control of the animal inoculated with the conventional vaccine, and selects the serum of the animal infected with the virus as a positive detection control. The results are shown in fig. 5, and all animals infected with wild type virus produced high titer antibodies against 3B protein 2H1 epitope (blocking rate is more than 75%) by 3B monoclonal antibody blocking ELISA detection; animals vaccinated with the commercial O/A bivalent inactivated vaccine, a portion of pigs (2/10), cattle (4/10) or sheep (4/10), produced antibodies (blocking rate greater than 50%) against the 3B protein 2H1 epitope; however, all animals vaccinated with the 3B deletion negative marker vaccine prepared by the present invention did not produce antibodies against the epitope of 3B protein 2H1 (blocking rate less than 25%). These results indicate that any 3B deletion-tagged FMDV strains constructed using the platform established in the present invention, including the modified virus rIRES obtained in the present invention_Bd43AB_m-PanAsia、rIRES_Bd43AB_mO/M98 and rIRES_Bd43AB_mthe-A/J14, when used as seed virus to produce foot-and-mouth disease inactivated vaccine for immunizing vaccinated animals, can realize the differential diagnosis of vaccinated animals and naturally infected animals.

SEQUENCE LISTING

<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)

<120> heat-resistant phenotype stable inheritance, negative mark carrying recombinant foot-and-mouth disease virus non-virulent strain and O/A type foot-and-mouth disease bivalent inactivated vaccine

<130> HLJ-2001-200315A

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 72

<212> DNA

<213> Artifical sequence

<400> 1

ggaccttacg aaggaccggt gaagaagcct gtcgctttga aagtaaaagc taagaacctg 60

attgtcactg aa 72

<210> 2

<211> 8019

<212> DNA

<213> Artifical sequence

<400> 2

ggatcctaat acgactcact ataggttgaa agggggcgtt agggtctcat ccctaacacg 60

ccaacgacag ctcctgcgct gcactctaca ctcacgtctg tgtgcgcgcg gggaccgttg 120

gactaccgtt cacccaccta cggttggact cacggcaccg cgcggccatt ttagctggat 180

tgtgtggacg aacgccactt gcgcactccg cgtgactggt taatactctt accactttcc 240

gcctacctgg tcgttggcgc tgtcctgggc actcccgttg ggggccgtcc ggtgctccac 300

ggtttccacg cgtgacaaac tacggtgatg gagccgcttc gtgcgagttg atcgcctggt 360

gtgtttcggc tgtcacccga agtccacctt tcaccccccc ccccccccca cctctcacaa 420

gtttttaccg cctttcccag cgttaaaggg aggtaaccac aagcttgcgt ctgtcttgct 480

cgacgataaa gggctgtgac cgcaagatga taccgccttt cccggcgtta attggatgta 540

accataagac gaaccttcac ccggaagtaa aacggcaaat tcgcatagtt ttgcccgttt 600

tcaggagaaa cgggacgtct gcgcacgaaa cgcgccgtcg cttgaggagg acttgtacaa 660

acacggtcca ttcaggtttc cacaaccgac acaaaccgtg caacttggaa ctccgcctgg 720

tctttccagg tctagagggg tgacattttg tactgtgctt gactccacgc tcggtccact 780

ggcgagtgct agtaacagca ctgttgcttc gtagcggagc atggtggccg cgggaactcc 840

tccttggtaa cagggacccg cggggccgaa agccacgtcc tcacggaccc accatgtgtg 900

caaccccagc acggcaactt tattgtgaaa accactttaa ggtgacactg atactggtac 960

tcaaccactg gtgacagcct aaggatgccc tccaggtacc ccggggtaac aagtgacacc 1020

cgggatctga ggaggggact actttacgta gtttaaaaaa cgtctaagct gaataggtga 1080

ccggaggccg gcacctttcc ttcgaacaac tgtctttaaa tgagcacaac tgactgtttc 1140

atcgctttgt tgtacgcttt cagagagatc aaaacactgt ttttatcacg aacgcgagga 1200

aagatggagt tcacacttca caacggtgag aagaaaacat tctactccag acccaacaac 1260

cacgacaact gctggttgaa cgccatcctc cagctgttta ggtacgttga tgaacctttc 1320

ttcgactggg tctactgttc acacgagaac ctcacactca atgctataaa acaattggaa 1380

gaaattactg gtctcgagct ccacgagggt ggaccacccg ctctcgttat ttggaacatc 1440

aaacacctgc tcaacaccgg aataggcacc gcttcgcgac ccagcgaagt gtgcatggta 1500

gacgggacgg acatgtgctt ggctgacttc catgctggca tcttcctgca aggacaggaa 1560

cacgctgtgt tcgcctgcgt cacctccaac gggtggtacg caatcgatga cgaggacttt 1620

tacccctgga cgccggaccc gtccgacgtt ctggtgttcg tcccgtacga ccaagaaccg 1680

ctcaacggag aatggaaaac aaaggttcag aaacgactgc agggtgccgg gcaatccagc 1740

ccggcgactg ggtcgcagaa ccagtcaggt aacactggaa gcattatcaa caattactac 1800

atgcagcagt accagaactc catggacaca caacttggtg acaacgctat tagtggaggc 1860

tccaacgagg ggtccacgga caccacctcc acccacacaa ccaacaccca aaacaacgac 1920

tggttttcaa agctagccag ttctgctttt agcggtcttt tcggcgctct tctcgctgac 1980

aagaaaaccg aggagaccac tcttcttgag gaccgcatcc tcactacccg caacgggcac 2040

acgacctcga caacccagtc aagcgttgga gtcacttacg ggtacgcgac agctgaggac 2100

tttgtgagcg gaccgaacac gtctgggctt gagaccaggg ttgtgcaggc agagcggttc 2160

ttcaaaaccc acttgttcga ctgggtcacc agtgacccgt tcggacggtg ctacctgctg 2220

gaactcccaa ctgaccacaa aggtgtctac ggtagcctaa ctgactctta tgcttacatg 2280

agaaacggtt gggatgtaga ggttactgca gtggggaatc agttcaacgg aggatgtctg 2340

ttggtggcta tggtaccaga actttgctct attgacaaga gagggcttta ccaactcacg 2400

ctcttccccc accagttcat caacccccgg acgaacatga cggcgcacat cactgtgcct 2460

tttgttggcg tcaaccgcta cgaccagtac aaggtacaca gaccttggac tctcgtggtc 2520

atggttgtgg ccccgctgac tgtcaacact gaaggtgccc cacagatcaa ggtttacgcc 2580

aacatcgccc ctactaacgt gcacgtcgcg ggtgagctcc cttctaagga agggatcttc 2640

cccgtggcat gtagcgacgg ttacggtggc ctggtgacca ctgacccaaa gacggctgac 2700

cccgcctacg ggaaagtgtt caatccacct cgcaacatgt tgccggggcg gttcaccaac 2760

ttccttgatg tggctgaggc gtgtcctacg tttctgcatt ttgagggtga cgtaccgtac 2820

gtgaccacaa agacggactc agacagggtg ctcgcccagt ttgacttgtc tctggcagca 2880

aaacacatgt caaacacctt cctggcaggt ctcgcccagt attacacaca gtacagcggc 2940

accatcaacc tgcacttcat gttcactgga cccactgacg cgaaagcgcg ttacatgatt 3000

gcatacgccc cccctggcat ggagccgccc aaaacacccg aggcggccgc tcactgcatt 3060

catgcggagt gggacacagg gttgaactca aaattcacat tttcaatccc ttacctttcg 3120

gcggctgact acgcgtacac cgcgtctgac tccgcggaga ccacaaacgt gcagggatgg 3180

gtttgcctgt ttcaaatcac acacgggaag gctgacggcg acgcgctggt cgttctagct 3240

agtgccggta aggactttga actgcgtttg ccagttgatg ctcgcacgca gaccacctct 3300

acaggtgagt cggctgaccc cgtaactgcc accgttgaga actacggtgg tgagacacag 3360

gtccagagac gccagcacac ggatgtctcg ttcatactag acagatttgt gaaagtaaca 3420

ccaaaagacc aaatcaatgt gttggacctg atgcaaaccc ctgcacacac tttggtaggc 3480

gcgctcctcc gtactgccac ttactacttt gcagatctag aagtggcagt gaaacacgag 3540

gggaacctta cctgggtccc gaatggggcg cccgaggcag cattggacaa caccaccaat 3600

ccaacggcct accacaaggc gccgctcacc cggcttgcac tgccttacac ggcaccacac 3660

cgtgtcttgg ctactgttta caacgggaac tgtaagtacg gcaagagccc cgtggccaac 3720

gcgagaggtg acctgcaagt gttgaccccg aaggcggcaa gaacgctgcc tacctccttc 3780

aattacggcg ccatcaaagc cactcgggtg actgaactgc tttaccgcat gaagagggcc 3840

gaaacgtact gcccccggcc tcttttggct attcacccga gcgaaactag acacaaacaa 3900

aagattgtgg cgcctgtgaa gcagctgcag aattttgatc tgctcaagct ggcaggagac 3960

gttgagtcca accctggacc cttcttcttc gctgacgtca ggtcaaattt ttccaagctg 4020

gttgagacca tcaaccaaat gcaggaggac atgtcaacaa aacacggacc cgactttaac 4080

cggttggtgt ctgcgtttga ggaactggcc gctggagtga gggctatcag gactggtctc 4140

gacgaggcca aaccctggta caagctcatc aagctactga gccgcctgtc atgcatggcc 4200

gctgtagcag cacggtcaaa ggacccagtc cttgtggcca tcatgctggc tgacaccggt 4260

ctcgagatcc tggacagtac ctttgtcgtg aagaagatct ccgactcgct ctccagtctc 4320

tttcacgtgc cggcccccgt cttcagtttc ggagccccga ttttgttggc cgggttggtc 4380

aaggtcgcct cgagtttctt ccggtccacg cccgaagacc ttgagagagc ggagaaacag 4440

ctcaaagcac gtgacatcaa tgacatattc gccattctca agaacggcga gtggctggtc 4500

aagctgatcc ttgccatccg cgactggatc aaggcatgga tcgcctcaga ggaaaagttt 4560

gtcaccatga cagacctggt gcccggtatc cttgaaaagc agcgggatct caacgaccca 4620

agcaagtacg aggaggccaa ggagtggctc gacaacgcgc gccaagcgtg tttgaagagc 4680

gggaacatcc acatcgcaaa cctttgcaaa gtggctgccc cagcacccag caggtcgagg 4740

cccgaacccg tggtcgtttg ccttcgtggc aaatcaggcc agggcaagag tttccttgcg 4800

aacgtgcttg cacaagcaat ttcaacccac ttcactggca gaaccgattc agtttggtac 4860

tgcccacctg accctgacca cttcgacggt tacaaccagc agaccgttgt agtaatggat 4920

gacctgggcc agaaccccga cgggaaggac tttaagtact tcgcccaaat ggtttcaact 4980

acggggttta tcccgcccat ggcttcactt gaggacaaag gcaaaccttt caacagcaag 5040

gtcatcatcg ccaccaccaa cctgtactcg ggcttcaccc cgagaactat ggtgtgccct 5100

gatgcgctga accggaggtt ccactttgac atcgacgtga gcgctaagga cggatacaaa 5160

attaacaaca aattggacat catcaaagct cttgaagaca cccacaccaa cccagtggca 5220

atgtttcaat acgactgtgc ccttctcaac ggcatggccg ttgaaatgaa gagaatgcaa 5280

caagacatgt tcaagcctca accgcccctc cagaacgtct accagcttgt tcaggaggtg 5340

attgaccggg tcgagctcca cgaaaaggtg tcgaaccacc cgatcttcaa gcagatctca 5400

attccttccc aaaaggctgt gctgtacttt ctcattgaga agggccagca cgaagcagca 5460

attgaattct ttgaggggat ggtgtgtgac tccattaagg aggagctccg gcccctaatc 5520

caacagacct catttgtgaa gcgcgctttt aagcgcctga aggaaaactt tgagattgtc 5580

gctctgtgtt tgaccctttt ggcgaacata gtgatcatga tccgcgggac tcgcaagaga 5640

caggaacccg tggaagggca accacaggct gaaggacctt acgaaggacc ggtgaagaag 5700

cctgtcgctt tgaaagtaaa agctaagaac ctgattgtca ctgaaggacc ttacgaagga 5760

ccggtgaaga aacctgtcgc tctgaaagtg aaagcaaaga acttgattgt cactgagagt 5820

ggtgctcccc cgactgactt gcaaaagatg gtcatgggca acaccaagcc tgttgagctc 5880

atcctcgacg ggaagacggt ggccatctgc tgcgccaccg gagtgtttgg taccgcctac 5940

cttgttcctc gccatctttt cgcagagaag tacgacaaga tcatgttgga cggcagagcc 6000

atgacagaca gtgactacag agtgtttgag tttgagatta aagtgaaagg gcaggacatg 6060

ctctcggacg ccgcgctcat ggtgctccac cgtgggaatc gcgtgcggga catcacgaag 6120

cacttccgtg atgtggcaag aatgaagaaa ggcacccccg tcgtcggcgt ggtcaacaac 6180

gctgatgttg ggagactgat cttctctggt gaggccctta cctacaagga cattgtagtg 6240

tgcatggacg gagacaccat gcccggtctc ttcgcctaca aagccgccac caaggcgggt 6300

tactgtggag gagccgttct tgcaaaggac ggagccgaga ctttcatcgt cggcactcac 6360

tccgcaggcg gcaacggagt tggctactgc tcgtgcgttt ccaggtctat gctgctaaaa 6420

atgaaggcac acatcgatcc cgaaccacac cacgagggat tgatagttga caccagagat 6480

gttgaggagc gcgtacatgt catgcgcaaa accaagctcg cacccaccgt ggcatacggt 6540

gtattcaacc ccgaatttgg gcctgccgcc ttgtccaacc aggacccgcg cctgaatgaa 6600

ggggttgtcc tcgatgaagt tatcttctct aaacacaagg aaaacacaaa gatgtctgag 6660

gaggacaaag cgctgttccg ccgctgtgct gctgactacg cgtcccgcct gcacagcgtg 6720

ctgggtacgg caaacgcccc actgagcatt tacgaggcaa ttaagggtgt cgacggactt 6780

gacgccatgg aaccagacac cgcgcctggc ctcccctggg ccctccaggg gaaacgccgt 6840

ggtgcgctca ttgacttcga gaacggcact gtcggacccg aggttgaggc tgctttgaag 6900

ctcatggaga aaagagagta caagtttgta tgccagacct ttctgaagga cgagatccgt 6960

ccgatggaaa aggtacgtgc cggtaagact cgcattgtcg acgtcctgcc tgttgaacac 7020

attctttaca ccaggatgat gattggtaga ttttgcgctc aaatgcactc aaacaacgga 7080

ccgcaaattg gttcggcggt tggttgtaat cctgatgttg attggcaaag atttggcacg 7140

cactttgctc agtacaaaaa cgtgtgggat gtggactatt cggcctttga cgccaaccac 7200

tgtagtgatg caatgaacat catgtttgag gaggtgttca acacggattt cggtttccac 7260

ccaaacgctg agtggatctt gaaaactctc gtggacactg aacacgccta tgagaacaaa 7320

cgcatcactg ttgaaggcgg gatgccgtct ggttgttccg cgacaagcat catcaacaca 7380

attttgaaca acatctacgt gctctacgcc ttgcgcagac actatgaggg agttgagctg 7440

gactcttaca ccatgatctc ctacggagac gacatcgtgg ttgcaagtga tcacgatctg 7500

gactttgagg ccctcaagcc tcacttcaaa tcccttggtc aaaccatcac tccagctgac 7560

aaaagtgaca aaggttttgt tcttggtcac tccattaccg atgtcacttt cctcaaaaga 7620

cacttccaca tggactatgg aactgggttt tacaaacctg tgatggcttc gaagaccctc 7680

gaggctatcc tctcctttgc acgtcgtggg accatacagg agaagttgat ctccgtggca 7740

ggactcgccg tccactctgg acctgatgag taccggcgtc tctttgagcc tttccagggc 7800

ctcttcgaga ttccaagcta cagatcactt tacctgcgtt gggtgaacgc cgtgtgcggt 7860

gacgcataat ccctcagatg tcacaactgg cagaaagacg ttgaggcgag cgacgccgta 7920

ggagtgaaaa gtccgaaagg gcttttcccg cttcctattt caaaaaaaaa aaaaaaaaaa 7980

aaaaaaaaaa aaaaaaaaaa aaaaaaagat atcgcatgc 8019

<210> 3

<211> 8018

<212> DNA

<213> Artifical sequence

<400> 3

ggatcctaat acgactcact ataggttgaa agggggcgtt agggtctcat ccctaacacg 60

ccaacgacag ctcctgcgct gcactctaca ctcacgtctg tgtgcgcgcg gggaccgttg 120

gactaccgtt cacccaccta cggttggact cacggcaccg cgcggccatt ttagctggat 180

tgtgtggacg aacgccactt gcgcactccg cgtgactggt taatactctt accactttcc 240

gcctacctgg tcgttggcgc tgtcctgggc actcccgttg ggggccgtcc ggtgctccac 300

ggtttccacg cgtgacaaac tacggtgatg gagccgcttc gtgcgagttg atcgcctggt 360

gtgtttcggc tgtcacccga agtccacctt tcaccccccc ccccccccca cctctcacaa 420

gtttttaccg cctttcccag cgttaaaggg aggtaaccac aagcttgcgt ctgtcttgct 480

cgacgataaa gggctgtgac cgcaagatga taccgccttt cccggcgtta attggatgta 540

accataagac gaaccttcac ccggaagtaa aacggcaaat tcgcatagtt ttgcccgttt 600

tcaggagaaa cgggacgtct gcgcacgaaa cgcgccgtcg cttgaggagg acttgtacaa 660

acacggtcca ttcaggtttc cacaaccgac acaaaccgtg caacttggaa ctccgcctgt 720

ctttccaggt ctagaggggt gacattttgt actgtgcttg actccacgct cggtccactg 780

gcgagtgcta gtaacagcac tgttgcttcg tagcggagca tggtggccgc gggaactcct 840

ccttggtaac agggacccgc ggggccgaaa gccacgtcct cacggaccca ccatgtgtgc 900

aaccccagca cggcaacttt attgtgaaaa ccactttaag gtgacactga tactggtact 960

caaccactgg tgacagccta aggatgccct ccaggtaccc cggggtaaca agtgacaccc 1020

gggatctgag gaggggacta ctttacgtag tttaaaaaac gtctaagctg aataggtgac 1080

cggaggccgg cacctttcct tcgaacaact gtctttaaat gagcacaact gactgtttca 1140

tcgctttgtt gtacgctttc agagagatca aaacactgtt tttatcacga acgcgaggaa 1200

agatggagtt cacacttcac aacggtgaga agaaaacatt ctactccaga cccaacaacc 1260

acgacaactg ctggttgaac gccatcctcc agctgtttag gtacgttgat gaacctttct 1320

tcgactgggt ctactgttca cacgagaacc tcacactcaa tgctataaaa caattggaag 1380

aaattactgg tctcgagctc cacgagggtg gaccacccgc tctcgttatt tggaacatca 1440

aacacctgct caacaccgga ataggcaccg cttcgcgacc cagcgaagtg tgcatggtag 1500

acgggacgga catgtgcttg gctgacttcc atgctggcat cttcctgcaa ggacaggaac 1560

acgctgtgtt cgcctgcgtc acctccaacg ggtggtacgc aatcgatgac gaggactttt 1620

acccctggac gccggacccg tccgacgttc tggtgttcgt cccgtacgac caagaaccgc 1680

tcaacggaga atggaaaaca aaggttcaga aacgactcag aggtgccggg caatccagcc 1740

cggcgactgg gtcgcagaac cagtcaggta acactggaag cattatcaac aattactaca 1800

tgcagcagta ccagaactcc atggacacac aacttggtga caacgctatt agtggaggct 1860

ccaacgaggg gtccacggac accacctcca cccacacaac caacacccaa aacaacgact 1920

ggttttcaaa gctagccagc tctgccttca gcggtctttt cggcgccctc ctcgccgata 1980

agaaaaccga ggagaccact cttctcgagg accgcatcct caccacccga aacggacaca 2040

ccacctcgac aacccagtcg agtgttggcg tcacgtacgg gtacgcaaca gctgaggatt 2100

ttgtgagcgg gccaaacacc tctggtcttg agaccagagt tgtccaggcg gaacggttct 2160

ttaaaaccca cctgttcgac tgggtcacca gtgatccgtt cggacggtgc cacttgttgg 2220

agctcccgac tgaccacaaa ggtgtctacg gctacctgac cgactcatac gcctacatga 2280

gaaacggttg ggatgttgag gtcaccgctg tggggaatca gttcaacgga ggctgcctac 2340

tggtggccat ggtgcctgaa ctttgttcca tcgacaagag agagctgtac cagcttacgc 2400

tcttccccca ccagttcatc aacccccgga cgaacatgac agcccacatc acggtgccct 2460

ttgttggcgt caaccgttac gatcagtaca aggtacacaa gccgtggacc cttgtggtta 2520

tggtcgtagc cccactgact gtcaacaccg aaggcgctcc gcagatcaag gtgtatgcca 2580

acatcgcacc caccaacgtg cacgtcgcgg gtgagttccc ttccaaagag gggattttcc 2640

ctgtggcctg tagcgacggt tatggcggct tggtgacaac tgacccaaag acggctgacc 2700

ccgtttacgg caaagtgttc aacccccccc gcaacatgtt gccggggcgg tttaccaacc 2760

tcctggacgt ggctgaggct tgccccacgt ttctgcactt cgatggtgac gtaccgtatg 2820

tgaccactaa gacggattcg gacagggtgc tcgcacaatt tgacttgtct ttggcagcaa 2880

aacacatgtc aaacaccttc cttgcaggtc ttgcccagta ctacacgcag tacagcggca 2940

ccgtcaacct gcacttcatg ttcacaggtc ccactgacgc gaaagcgcgt tacatgattg 3000

cgtatgcccc tccgggcatg gagccgccta aaacacctga ggcggccgct cactgcattc 3060

acgcagagtg ggacacgggt ctgaactcaa agtttacctt ttccatcccc tacctctcgg 3120

cggctgatta cgcgtacacc gcgtctgacg ctgctgagac cacaaatgtt cagggatggg 3180

tctgcttatt tcaaataaca cacgggaaag ctgagggtga cgctcttgtc gtgatggcca 3240

gtgctggcaa agactttgag ctgcgcctgc ctgtagacgc tcggcagcag accacttcga 3300

caggcgagtc ggctgacccc gtgactgcca ccgttgagga ctacggcggc gagacacagg 3360

tccagaggcg ccaccacaca gacgtctcat tcatattgga cagatttgtg aaagtcacac 3420

caaaagactc aataaatgta ttggacctga tgcagacccc ctcccacacc ctagtagggg 3480

cgctcctccg cactgccact tactatttcg ctgatctaga ggtggcagtg aaacacgagg 3540

gggaccttac ctgggtgcca aatggagcac ctgaagcagc cttggacaac accaccaacc 3600

caacggcgta ccataaggcg ccgcttaccc ggcttgcatt gccctacacg gcaccacacc 3660

gtgttttggc caccgtttac aacgggaact gcaaatacgc cgggggctca ctgcccaacg 3720

tgagaggcga tctccaagtg ctggctcaga aggcagcgag gccgctgcct acttctttca 3780

actacggtgc catcaaagcc actcgggtga cagaactgct gtaccgcatg aagagggccg 3840

agacgtactg tcctcggccc ctcttggctg ttcacccgag tgcggccaga cacaaacaga 3900

aaatagtggc acctgtaaag cagcttttga attttgatct gctcaagctg gcaggagacg 3960

ttgagtccaa ccctggaccc ttcttcttcg ctgacgtcag gtcaaatttt tccaagctgg 4020

ttgagaccat caaccaaatg caggaggaca tgtcaacaaa acacggaccc gactttaacc 4080

ggttggtgtc tgcgtttgag gaactggccg ctggagtgag ggctatcagg actggtctcg 4140

acgaggccaa accctggtac aagctcatca agctactgag ccgcctgtca tgcatggccg 4200

ctgtagcagc acggtcaaag gacccagtcc ttgtggccat catgctggct gacaccggtc 4260

tcgagatcct ggacagtacc tttgtcgtga agaagatctc cgactcgctc tccagtctct 4320

ttcacgtgcc ggcccccgtc ttcagtttcg gagccccgat tttgttggcc gggttggtca 4380

aggtcgcctc gagtttcttc cggtccacgc ccgaagacct tgagagagcg gagaaacagc 4440

tcaaagcacg tgacatcaat gacatattcg ccattctcaa gaacggcgag tggctggtca 4500

agctgatcct tgccatccgc gactggatca aggcatggat cgcctcagag gaaaagtttg 4560

tcaccatgac agacctggtg cccggtatcc ttgaaaagca gcgggatctc aacgacccaa 4620

gcaagtacga ggaggccaag gagtggctcg acaacgcgcg ccaagcgtgt ttgaagagcg 4680

ggaacatcca catcgcaaac ctttgcaaag tggctgcccc agcacccagc aggtcgaggc 4740

ccgaacccgt ggtcgtttgc cttcgtggca aatcaggcca gggcaagagt ttccttgcga 4800

acgtgcttgc acaagcaatt tcaacccact tcactggcag aaccgattca gtttggtact 4860

gcccacctga ccctgaccac ttcgacggtt acaaccagca gaccgttgta gtaatggatg 4920

acctgggcca gaaccccgac gggaaggact ttaagtactt cgcccaaatg gtttcaacta 4980

cggggtttat cccgcccatg gcttcacttg aggacaaagg caaacctttc aacagcaagg 5040

tcatcatcgc caccaccaac ctgtactcgg gcttcacccc gagaactatg gtgtgccctg 5100

atgcgctgaa ccggaggttc cactttgaca tcgacgtgag cgctaaggac ggatacaaaa 5160

ttaacaacaa attggacatc atcaaagctc ttgaagacac ccacaccaac ccagtggcaa 5220

tgtttcaata cgactgtgcc cttctcaacg gcatggccgt tgaaatgaag agaatgcaac 5280

aagacatgtt caagcctcaa ccgcccctcc agaacgtcta ccagcttgtt caggaggtga 5340

ttgaccgggt cgagctccac gaaaaggtgt cgaaccaccc gatcttcaag cagatctcaa 5400

ttccttccca aaaggctgtg ctgtactttc tcattgagaa gggccagcac gaagcagcaa 5460

ttgaattctt tgaggggatg gtgtgtgact ccattaagga ggagctccgg cccctaatcc 5520

aacagacctc atttgtgaag cgcgctttta agcgcctgaa ggaaaacttt gagattgtcg 5580

ctctgtgttt gacccttttg gcgaacatag tgatcatgat ccgcgggact cgcaagagac 5640

aggaacccgt ggaagggcaa ccacaggctg aaggacctta cgaaggaccg gtgaagaagc 5700

ctgtcgcttt gaaagtaaaa gctaagaacc tgattgtcac tgaaggacct tacgaaggac 5760

cggtgaagaa acctgtcgct ctgaaagtga aagcaaagaa cttgattgtc actgagagtg 5820

gtgctccccc gactgacttg caaaagatgg tcatgggcaa caccaagcct gttgagctca 5880

tcctcgacgg gaagacggtg gccatctgct gcgccaccgg agtgtttggt accgcctacc 5940

ttgttcctcg ccatcttttc gcagagaagt acgacaagat catgttggac ggcagagcca 6000

tgacagacag tgactacaga gtgtttgagt ttgagattaa agtgaaaggg caggacatgc 6060

tctcggacgc cgcgctcatg gtgctccacc gtgggaatcg cgtgcgggac atcacgaagc 6120

acttccgtga tgtggcaaga atgaagaaag gcacccccgt cgtcggcgtg gtcaacaacg 6180

ctgatgttgg gagactgatc ttctctggtg aggcccttac ctacaaggac attgtagtgt 6240

gcatggacgg agacaccatg cccggtctct tcgcctacaa agccgccacc aaggcgggtt 6300

actgtggagg agccgttctt gcaaaggacg gagccgagac tttcatcgtc ggcactcact 6360

ccgcaggcgg caacggagtt ggctactgct cgtgcgtttc caggtctatg ctgctaaaaa 6420

tgaaggcaca catcgatccc gaaccacacc acgagggatt gatagttgac accagagatg 6480

ttgaggagcg cgtacatgtc atgcgcaaaa ccaagctcgc acccaccgtg gcatacggtg 6540

tattcaaccc cgaatttggg cctgccgcct tgtccaacca ggacccgcgc ctgaatgaag 6600

gggttgtcct cgatgaagtt atcttctcta aacacaagga aaacacaaag atgtctgagg 6660

aggacaaagc gctgttccgc cgctgtgctg ctgactacgc gtcccgcctg cacagcgtgc 6720

tgggtacggc aaacgcccca ctgagcattt acgaggcaat taagggtgtc gacggacttg 6780

acgccatgga accagacacc gcgcctggcc tcccctgggc cctccagggg aaacgccgtg 6840

gtgcgctcat tgacttcgag aacggcactg tcggacccga ggttgaggct gctttgaagc 6900

tcatggagaa aagagagtac aagtttgtat gccagacctt tctgaaggac gagatccgtc 6960

cgatggaaaa ggtacgtgcc ggtaagactc gcattgtcga cgtcctgcct gttgaacaca 7020

ttctttacac caggatgatg attggtagat tttgcgctca aatgcactca aacaacggac 7080

cgcaaattgg ttcggcggtt ggttgtaatc ctgatgttga ttggcaaaga tttggcacgc 7140

actttgctca gtacaaaaac gtgtgggatg tggactattc ggcctttgac gccaaccact 7200

gtagtgatgc aatgaacatc atgtttgagg aggtgttcaa cacggatttc ggtttccacc 7260

caaacgctga gtggatcttg aaaactctcg tggacactga acacgcctat gagaacaaac 7320

gcatcactgt tgaaggcggg atgccgtctg gttgttccgc gacaagcatc atcaacacaa 7380

ttttgaacaa catctacgtg ctctacgcct tgcgcagaca ctatgaggga gttgagctgg 7440

actcttacac catgatctcc tacggagacg acatcgtggt tgcaagtgat cacgatctgg 7500

actttgaggc cctcaagcct cacttcaaat cccttggtca aaccatcact ccagctgaca 7560

aaagtgacaa aggttttgtt cttggtcact ccattaccga tgtcactttc ctcaaaagac 7620

acttccacat ggactatgga actgggtttt acaaacctgt gatggcttcg aagaccctcg 7680

aggctatcct ctcctttgca cgtcgtggga ccatacagga gaagttgatc tccgtggcag 7740

gactcgccgt ccactctgga cctgatgagt accggcgtct ctttgagcct ttccagggcc 7800

tcttcgagat tccaagctac agatcacttt acctgcgttg ggtgaacgcc gtgtgcggtg 7860

acgcataatc cctcagatgt cacaactggc agaaagacgt tgaggcgagc gacgccgtag 7920

gagtgaaaag tccgaaaggg cttttcccgc ttcctatttc aaaaaaaaaa aaaaaaaaaa 7980

aaaaaaaaaa aaaaaaaaaa aaaaaagata tcgcatgc 8018

<210> 4

<211> 8019

<212> DNA

<213> Artifical sequence

<400> 4

ggatcctaat acgactcact ataggttgaa agggggcgtt agggtctcat ccctaacacg 60

ccaacgacag ctcctgcgct gcactctaca ctcacgtctg tgtgcgcgcg gggaccgttg 120

gactaccgtt cacccaccta cggttggact cacggcaccg cgcggccatt ttagctggat 180

tgtgtggacg aacgccactt gcgcactccg cgtgactggt taatactctt accactttcc 240

gcctacctgg tcgttggcgc tgtcctgggc actcccgttg ggggccgtcc ggtgctccac 300

ggtttccacg cgtgacaaac tacggtgatg gagccgcttc gtgcgagttg atcgcctggt 360

gtgtttcggc tgtcacccga agtccacctt tcaccccccc ccccccccca cctctcacaa 420

gtttttaccg cctttcccag cgttaaaggg aggtaaccac aagcttgcgt ctgtcttgct 480

cgacgataaa gggctgtgac cgcaagatga taccgccttt cccggcgtta attggatgta 540

accataagac gaaccttcac ccggaagtaa aacggcaaat tcgcatagtt ttgcccgttt 600

tcaggagaaa cgggacgtct gcgcacgaaa cgcgccgtcg cttgaggagg acttgtacaa 660

acacggtcca ttcaggtttc cacaaccgac acaaaccgtg caacttggaa ctccgcctgg 720

tctttccagg tctagagggg tgacattttg tactgtgctt gactccacgc tcggtccact 780

ggcgagtgct agtaacagca ctgttgcttc gtagcggagc atggtggccg cgggaactcc 840

tccttggtaa cagggacccg cggggccgaa agccacgtcc tcacggaccc accatgtgtg 900

caaccccagc acggcaactt tattgtgaaa accactttaa ggtgacactg atactggtac 960

tcaaccactg gtgacagcct aaggatgccc tccaggtacc ccggggtaac aagtgacacc 1020

cgggatctga ggaggggact actttacgta gtttaaaaaa cgtctaagct gaataggtga 1080

ccggaggccg gcacctttcc ttcgaacaac tgtctttaaa tgagcacaac tgactgtttc 1140

atcgctttgt tgtacgcttt cagagagatc aaaacactgt ttttatcacg aacgcgagga 1200

aagatggagt tcacacttca caacggtgag aagaaaacat tctactccag acccaacaac 1260

cacgacaact gctggttgaa cgccatcctc cagctgttta ggtacgttga tgaacctttc 1320

ttcgactggg tctactgttc acacgagaac ctcacactca atgctataaa acaattggaa 1380

gaaattactg gtctcgagct ccacgagggt ggaccacccg ctctcgttat ttggaacatc 1440

aaacacctgc tcaacaccgg aataggcacc gcttcgcgac ccagcgaagt gtgcatggta 1500

gacgggacgg acatgtgctt ggctgacttc catgctggca tcttcctgca aggacaggaa 1560

cacgctgtgt tcgcctgcgt cacctccaac gggtggtacg caatcgatga cgaggacttt 1620

tacccctgga cgccggaccc gtccgacgtt ctggtgttcg tcccgtacga ccaagaaccg 1680

ctcaacggag aatggaaaac aaaggttcag aaacgactca gaggtgccgg gcaatccagc 1740

ccggcgactg ggtcgcagaa ccagtcaggt aacactggaa gcattatcaa caattactac 1800

atgcagcagt accagaactc catggacaca caacttggtg acaacgctat tagtggaggc 1860

tccaacgagg ggtccacgga caccacctcc acccacacaa ccaacaccca aaacaacgac 1920

tggttttcaa agctggcaag ttccgccttc accgggcttt tcggcgcact gctcgccgac 1980

aaaaagaccg aagagacaac tctcctggag gaccgcatcc tcaccactcg caatggacac 2040

accacctcca caactcaatc gagtgtgggg gtcacctgcg gatattcaac tggtgaagac 2100

cacgtttctg ggcctaacac atcaggtttg gagacgcggg tggtgcaggc tgagaggttt 2160

ttcaagaagc acttgtttga ctggacaacg gacaaaccct ttggtcacat tgaaaaattg 2220

gaacttccca ctgaacacaa aggtgtctac ggacagctgg tagaatcctt tgcatacatg 2280

agaaatggct gggacgtgga ggtgtctgct gttggcaacc agttcaacgg cgggtgcctt 2340

ctcgtggcca tgatacccga gttcaaagag ttcacccagc gtgagaaata ccagctcacc 2400

ttgttcccac accagtttat cagccccaga accaacatga ctgcgcacat cacggtcccg 2460

taccttggtg tgaacagata tgaccagtac aagaaacaca aaccctggac gttggtggtg 2520

atggtggtct caccacttac cactagctcc attggtgcaa cacagatcaa ggtctacgcc 2580

aacatcgccc cgacccacgt acacgtggcc ggcgagctcc cctcgaaaga ggggatcgtg 2640

ccggtcgctt gctcggacgg gtacggtggc ctggtgacaa cagaccccaa aacagctgac 2700

cctgcttacg gtatggtgta caacccaccc aggaccaatt accctgggcg gtttacaaat 2760

ttgttggatg tggcagaggc gtgccccacc ttcctctgtt tcgacgacgg gaaaccgtat 2820

atcgtgacaa ggacggacga gcaacgcctc ttagccaagt ttgacctctc tcttgctgca 2880

aagcacatgt caaacaccta cctgtcaggg ctagcacagt actacgcaca gtactctggc 2940

accatcaatt tgcacttcat gtttactggt tccactgact caaaggcccg ctacatggtg 3000

gcgtacgttc cacccggagt ggaaacgcca ccggacacgc ctgagaaggc tgcacactgc 3060

atccacgcag aatgggacac gggcctaaat tccaaattca ccttttcaat cccgtacgta 3120

tctgctgcag actacgcgta cacagcgtct gacgaggcag aaacgacaaa cgtacaggga 3180

tgggtttgca tctaccaaat tacccacggg aaggccgaac aagacactct ggtcgtgtcg 3240

gttagcgccg gcaaagactt cgagctgcgc ctccccattg acccccgtgc gcaaaccacc 3300

accaccgggg aatcagcaga ccctgtcaca accaccgttg agcactacgg tggcgagaca 3360

caagtacagc ggcgttacca caccgacgtc ggcttcttaa tggacaggtt cgtgcagatc 3420

aagcctgtgg gccccacaca tgtcattgac ctcatgcaga cacaccaaca cgggctggtg 3480

ggcgctatgt tgcgcgcggc cacctactac ttttctgatc ttgagattgt ggtgaaccac 3540

acgggtaacc taacgtgggt acccaatgga gcacccgagg cagcactgca aaacacgagc 3600

aaccccactg cttaccacaa agcgccgttc acgaggcttg cgctccccta caccgcgcca 3660

caccgcgtgc tggcaactgt gtacagtggg acgagcaagt actccgcacc tcaaaaccgg 3720

cgaggtgact tgggtcctct cgcggcgaga ctcgctgcac agctccctgc ctccttcaac 3780

ttcggtgcaa ttcgggccac ggagatccgc gaactccttg tgcgcatgaa gcgcgccgag 3840

ctctactgcc ccaggccgct gttggcggtg gaggtgtcgt cgcaagacag acacaagcag 3900

aaaatcattg cccctgcaaa gcaacttttg aattttgatc tgctcaagct ggcaggagac 3960

gttgagtcca accctggacc cttcttcttc gctgacgtca ggtcaaattt ttccaagctg 4020

gttgagacca tcaaccaaat gcaggaggac atgtcaacaa aacacggacc cgactttaac 4080

cggttggtgt ctgcgtttga ggaactggcc gctggagtga gggctatcag gactggtctc 4140

gacgaggcca aaccctggta caagctcatc aagctactga gccgcctgtc atgcatggcc 4200

gctgtagcag cacggtcaaa ggacccagtc cttgtggcca tcatgctggc tgacaccggt 4260

ctcgagatcc tggacagtac ctttgtcgtg aagaagatct ccgactcgct ctccagtctc 4320

tttcacgtgc cggcccccgt cttcagtttc ggagccccga ttttgttggc cgggttggtc 4380

aaggtcgcct cgagtttctt ccggtccacg cccgaagacc ttgagagagc ggagaaacag 4440

ctcaaagcac gtgacatcaa tgacatattc gccattctca agaacggcga gtggctggtc 4500

aagctgatcc ttgccatccg cgactggatc aaggcatgga tcgcctcaga ggaaaagttt 4560

gtcaccatga cagacctggt gcccggtatc cttgaaaagc agcgggatct caacgaccca 4620

agcaagtacg aggaggccaa ggagtggctc gacaacgcgc gccaagcgtg tttgaagagc 4680

gggaacatcc acatcgcaaa cctttgcaaa gtggctgccc cagcacccag caggtcgagg 4740

cccgaacccg tggtcgtttg ccttcgtggc aaatcaggcc agggcaagag tttccttgcg 4800

aacgtgcttg cacaagcaat ttcaacccac ttcactggca gaaccgattc agtttggtac 4860

tgcccacctg accctgacca cttcgacggt tacaaccagc agaccgttgt agtaatggat 4920

gacctgggcc agaaccccga cgggaaggac tttaagtact tcgcccaaat ggtttcaact 4980

acggggttta tcccgcccat ggcttcactt gaggacaaag gcaaaccttt caacagcaag 5040

gtcatcatcg ccaccaccaa cctgtactcg ggcttcaccc cgagaactat ggtgtgccct 5100

gatgcgctga accggaggtt ccactttgac atcgacgtga gcgctaagga cggatacaaa 5160

attaacaaca aattggacat catcaaagct cttgaagaca cccacaccaa cccagtggca 5220

atgtttcaat acgactgtgc ccttctcaac ggcatggccg ttgaaatgaa gagaatgcaa 5280

caagacatgt tcaagcctca accgcccctc cagaacgtct accagcttgt tcaggaggtg 5340

attgaccggg tcgagctcca cgaaaaggtg tcgaaccacc cgatcttcaa gcagatctca 5400

attccttccc aaaaggctgt gctgtacttt ctcattgaga agggccagca cgaagcagca 5460

attgaattct ttgaggggat ggtgtgtgac tccattaagg aggagctccg gcccctaatc 5520

caacagacct catttgtgaa gcgcgctttt aagcgcctga aggaaaactt tgagattgtc 5580

gctctgtgtt tgaccctttt ggcgaacata gtgatcatga tccgcgggac tcgcaagaga 5640

caggaacccg tggaagggca accacaggct gaaggacctt acgaaggacc ggtgaagaag 5700

cctgtcgctt tgaaagtaaa agctaagaac ctgattgtca ctgaaggacc ttacgaagga 5760

ccggtgaaga aacctgtcgc tctgaaagtg aaagcaaaga acttgattgt cactgagagt 5820

ggtgctcccc cgactgactt gcaaaagatg gtcatgggca acaccaagcc tgttgagctc 5880

atcctcgacg ggaagacggt ggccatctgc tgcgccaccg gagtgtttgg taccgcctac 5940

cttgttcctc gccatctttt cgcagagaag tacgacaaga tcatgttgga cggcagagcc 6000

atgacagaca gtgactacag agtgtttgag tttgagatta aagtgaaagg gcaggacatg 6060

ctctcggacg ccgcgctcat ggtgctccac cgtgggaatc gcgtgcggga catcacgaag 6120

cacttccgtg atgtggcaag aatgaagaaa ggcacccccg tcgtcggcgt ggtcaacaac 6180

gctgatgttg ggagactgat cttctctggt gaggccctta cctacaagga cattgtagtg 6240

tgcatggacg gagacaccat gcccggtctc ttcgcctaca aagccgccac caaggcgggt 6300

tactgtggag gagccgttct tgcaaaggac ggagccgaga ctttcatcgt cggcactcac 6360

tccgcaggcg gcaacggagt tggctactgc tcgtgcgttt ccaggtctat gctgctaaaa 6420

atgaaggcac acatcgatcc cgaaccacac cacgagggat tgatagttga caccagagat 6480

gttgaggagc gcgtacatgt catgcgcaaa accaagctcg cacccaccgt ggcatacggt 6540

gtattcaacc ccgaatttgg gcctgccgcc ttgtccaacc aggacccgcg cctgaatgaa 6600

ggggttgtcc tcgatgaagt tatcttctct aaacacaagg aaaacacaaa gatgtctgag 6660

gaggacaaag cgctgttccg ccgctgtgct gctgactacg cgtcccgcct gcacagcgtg 6720

ctgggtacgg caaacgcccc actgagcatt tacgaggcaa ttaagggtgt cgacggactt 6780

gacgccatgg aaccagacac cgcgcctggc ctcccctggg ccctccaggg gaaacgccgt 6840

ggtgcgctca ttgacttcga gaacggcact gtcggacccg aggttgaggc tgctttgaag 6900

ctcatggaga aaagagagta caagtttgta tgccagacct ttctgaagga cgagatccgt 6960

ccgatggaaa aggtacgtgc cggtaagact cgcattgtcg acgtcctgcc tgttgaacac 7020

attctttaca ccaggatgat gattggtaga ttttgcgctc aaatgcactc aaacaacgga 7080

ccgcaaattg gttcggcggt tggttgtaat cctgatgttg attggcaaag atttggcacg 7140

cactttgctc agtacaaaaa cgtgtgggat gtggactatt cggcctttga cgccaaccac 7200

tgtagtgatg caatgaacat catgtttgag gaggtgttca acacggattt cggtttccac 7260

ccaaacgctg agtggatctt gaaaactctc gtggacactg aacacgccta tgagaacaaa 7320

cgcatcactg ttgaaggcgg gatgccgtct ggttgttccg cgacaagcat catcaacaca 7380

attttgaaca acatctacgt gctctacgcc ttgcgcagac actatgaggg agttgagctg 7440

gactcttaca ccatgatctc ctacggagac gacatcgtgg ttgcaagtga tcacgatctg 7500

gactttgagg ccctcaagcc tcacttcaaa tcccttggtc aaaccatcac tccagctgac 7560

aaaagtgaca aaggttttgt tcttggtcac tccattaccg atgtcacttt cctcaaaaga 7620

cacttccaca tggactatgg aactgggttt tacaaacctg tgatggcttc gaagaccctc 7680

gaggctatcc tctcctttgc acgtcgtggg accatacagg agaagttgat ctccgtggca 7740

ggactcgccg tccactctgg acctgatgag taccggcgtc tctttgagcc tttccagggc 7800

ctcttcgaga ttccaagcta cagatcactt tacctgcgtt gggtgaacgc cgtgtgcggt 7860

gacgcataat ccctcagatg tcacaactgg cagaaagacg ttgaggcgag cgacgccgta 7920

ggagtgaaaa gtccgaaagg gcttttcccg cttcctattt caaaaaaaaa aaaaaaaaaa 7980

aaaaaaaaaa aaaaaaaaaa aaaaaaagat atcgcatgc 8019

Claims (13)

1. FMDV full-length cDNA infectious clone plasmid, characterized in that, the domain 4 of IRES of FMDV genome is replaced by the domain 4 of IRES of bovine rhinovirus, the coding region of 84-143 amino acids of FMDV non-structural protein 3A is deleted, the coding region of FMDV 3B1 and 3B2 proteins is replaced by the coding region of FMDV 3B3 protein, and Pst I enzyme cutting site is introduced at two sides of FMDV structural protein P1 coding region; the nucleotide sequence of the coding region of the 3B3 protein of FMDV is shown as SEQ ID NO. 1.
2. 2. An FMDV full-length cDNA infectious clone plasmid according to claim 1, characterized in that its nucleotide sequence is shown in SEQ ID No. 2.
3. 3. The recombinant FMDV non-virulent strain carrying the negative marker rescued by the FMDV full-length cDNA infectious clone plasmid of claim 1 or 2 through a reverse genetic method.
4. An infectious cloning plasmid of FMDV full-length cDNA of type O, characterized in that the structural protein P1 coding region of FMDV full-length cDNA infectious cloning plasmid of claim 1 is replaced by the structural protein P1 coding region of FMDV; wherein, the 79 th tyrosine of the structural protein VP2 of the O-type FMDV is mutated into histidine, and the 93 th serine is mutated into tyrosine.
5. 5. An infectious full-length cDNA cloning plasmid of FMDV type O according to claim 4, wherein the nucleotide sequence is shown as SEQ ID No. 3.
6. 6. The O-type FMDV infectious full-length cDNA clone plasmid of claim 4 or 5 is used for rescuing the obtained recombinant O-type foot-and-mouth disease virus avirulent strain with a heat-resistant phenotype and a 3B epitope deletion negative marker by a reverse genetic method.
7. An FMDV full-length cDNA infectious clone plasmid type A, characterized in that the structural protein P1 coding region of FMDV full-length cDNA infectious clone plasmid of claim 1 is replaced by the structural protein P1 coding region of FMDV type A; wherein, alanine at position 3 of structural protein VP1 of A type FMDV is mutated into threonine and asparagine at position 17 is mutated into histidine.
8. 8. An infectious full-length cDNA cloning plasmid of FMDV type A according to claim 7, wherein the nucleotide sequence is represented by SEQ ID No. 4.
9. 9. The recombinant A-type foot-and-mouth disease virus avirulent strain with heat-resistant phenotype and 3B epitope deletion negative marker obtained by rescuing the A-type FMDV full-length cDNA infectious clone plasmid of claim 7 or 8 by reverse genetic method.
10. 10. The bivalent inactivated vaccine for preventing and treating O-type foot-and-mouth disease and A-type foot-and-mouth disease is characterized by comprising the recombinant O-type foot-and-mouth disease virus avirulent strain of claim 6, the recombinant A-type foot-and-mouth disease virus avirulent strain of claim 9 and an immunologic adjuvant.
11. 11. The use of the recombinant FMDV avirulent strain according to claim 3 in the preparation of drugs for the prevention and treatment of foot and mouth disease or in the preparation of reagents for the differential diagnosis of foot and mouth disease vaccinated animals and naturally infected animals.
12. 12. The use of the recombinant aftosa virus avirulent strain of claim 6 in the preparation of a medicament for the prevention and treatment of aftosa, or in the preparation of a reagent for differential diagnosis of aftosa-vaccinated animals and naturally-infected animals.
13. 13. The use of the recombinant aftosa virus avirulent strain of claim 9 in the preparation of a medicament for the prevention and treatment of aftosa, or in the preparation of a reagent for differential diagnosis of aftosa-vaccinated animals and naturally-infected animals.

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