CN101948811A - Method for expanding antigen spectrum of foot-and-mouth disease vaccine strain by reverse genetic operation and preparation method of vaccine - Google Patents
Method for expanding antigen spectrum of foot-and-mouth disease vaccine strain by reverse genetic operation and preparation method of vaccine Download PDFInfo
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Abstract
The invention relates to a method for expanding the antigen spectrum of a foot-and-mouth disease vaccine strain by reverse genetic operation and a preparation method of a vaccine. The amino acid sequence of the VP3 and VP1 structural proteins of the foot-and-mouth disease virus strain of the invention is represented by the amino acid residues from a position 304 to a position 736 in SEQ ID No.4. Experiments show that the vaccine prepared from the mutant virus strain obtained by the invention can resist porcine epidemic viruses of China O/TL/Taiwan/97 lineage, Pan-Asia O/China/99 lineage and Southeast Asia Myanmar O/GS/2010/98 lineage, has a characteristic of wide antigen spectrum, can immunize pigs and obviously improve the rate of protection against foot-and-mouth disease viruses which are of the same type and have antigenicity difference, achieves an immune effect of cross protection, and is expected to play an important role in the prevention and control of foot-and-mouth disease.
Description
Technical field
The present invention relates to utilize reverse genetic manipulation to expand aftosa vaccine strain spectrotype and vaccine production method.
Background technology
(foot-and-mouth disease is by foot and mouth disease virus (foot-and-mouth disease virus, multiple a kind of acute, hot, height contagious disease artiodactylous such as the pig that FMDV) causes, ox, sheep FMD) to foot and mouth disease.Extremely strong because of its infectivity, OIE (FAO/OIE) classifies it as and must report first of the eqpidemic disease, and China is defined as a class zoonosis.The outburst of this disease and the popular productivity of domestic animal that makes descend, and live poultry and Livestock Product Trade are stagnated, and financial loss is huge; Simultaneously, cause serious negative impact for public health and national reputation.Therefore, countries in the world are attached great importance to these sick prevention and control.At present, many in the world countries, especially Africa, Asia and south American countries, this disease frequency is frequent, and is popular serious.The popularity outburst in the whole world appears in addition, frequently.
A major measure of prevention and control foot and mouth disease is to use the vaccine immunity susceptible animal.Effective vaccine commonly used is an inactivated vaccine.Its preparation is the cell with the foot and mouth disease virus inoculation culture of screening, a large amount of virus of proliferation, and results virus forms with adjuvant emulsion after deactivation.The foot and mouth disease virus of screening is vaccine strain, also is kind of a poison.The requirement that vaccine strain must satisfy is that its antigen relation is close with popular foot and mouth disease virus (popular poison).Yet, foot and mouth disease virus easily morphs, plant the difference poultry, different areas popular virus, through repeated infection, it is popular to circulate, can derivation go out a plurality of variants (the close strain of genetic affinity is classified as a pedigree), variant [the B.Borrego of antigen, I.S.Novella, E.Giralt, D.Andreu, E.Domingo.1993.Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection.J.Virol.67,6071-6079, A.Holguin, J.Hernandez, M.A.Martinez, M.G.Mateu, E.Domingo.1997.Differential restrictions on antigenic variation among antigenic sites of foot-and-mouth disease virus in the absence of antibody selection.J Gene Virol., 78:601-609].Its result weakens the validity of original vaccine strain even loses, and is difficult to reach immunity epidemic prevention requirement, and the vaccine strain that need more renew just can play effective immune prevention and control effect.
The basic reason that vaccine strain validity weakens is that antigen relation and popular poison are become estranged, and does not match.The same with other virus, antigen relation between the different virus strain is relevant with the aminoacid sequence of forming viral protein (structural protein), and (structural protein of foot and mouth disease virus have VP4, VP2, VP3, four kinds of VP1, wherein VP1 is exposed to the virion surface, and has one section amino acid chain formation ring texture (G-H ring) to expose).The molecular basis of the decision antigen relation that more speaks by the book is the similarities and differences that antigen decision family or antigen site aminoacid sequence are formed.When determining family or antigen site aminoacid sequence, antigen morphs, form the discrepant variant of antigenicity, when many places variation or amino acid accumulation variation take place, the variant of this derivation and proviral antigen relation are become estranged gradually, provirus is difficult to bring into play effective immunoprophylactic effect as vaccine strain, needs the new vaccine strain of screening.Waste time and energy but screen new vaccine strain, even screening is less than the kind poison that meets the demands.More difficult is, in an area or country have the popular poison of a plurality of antigenic differences, screen a vaccine strain that can mate the popular poison of a plurality of pedigrees and may realize hardly.
Summary of the invention
An object of the present invention is to provide a strain hoof-and-mouth disease poison strain.
Hoof-and-mouth disease poison strain provided by the present invention, the aminoacid sequence of its VP3 and VP1 structural protein is shown in 304-736 amino acids residue among the SEQ ID NO:4.
The structural protein of above-mentioned hoof-and-mouth disease poison strain are shown in 1-736 amino acids residue among the SEQ ID NO:4;
In the above-mentioned hoof-and-mouth disease poison strain, the encoding gene of described VP3 and VP1 structural protein is shown in 2569-3867 position Nucleotide among the SEQ ID NO:2;
In the above-mentioned hoof-and-mouth disease poison strain, the encoding gene of structural protein is shown in 1660-3867 position Nucleotide among the SEQ ID NO:2.
The cDNA sequence of the geneome RNA correspondence of above-mentioned hoof-and-mouth disease poison strain is shown in SEQ ID NO:2.
Another object of the present invention provides a kind of vaccine.
Vaccine provided by the present invention, its activeconstituents are the above-mentioned arbitrary described hoof-and-mouth disease poison strain of deactivation.
In the above-mentioned vaccine, described vaccine is an aftosa vaccine;
In the above-mentioned vaccine, the adjuvant that uses is IS201; The name of product of adjuvant IS201 is Montanide ISA 201VG, and available from French SEPPIC company, catalog number is L01408.
In the above-mentioned vaccine, described foot and mouth disease is the foot and mouth disease that O type foot and mouth disease virus causes;
In the above-mentioned vaccine, described O type foot and mouth disease virus is the pig source;
In the above-mentioned vaccine, described O type foot and mouth disease virus is Chinese pedigree or Pan Asia pedigree or Burma's 98 pedigrees;
In the above-mentioned vaccine, described Chinese pedigree virus stain is O/TL/Taiwan/97, and described Pan Asia pedigree virus stain is O/China/99, and described Burma 98 pedigree virus stains are O/GS/2010.
The application of above-mentioned arbitrary described hoof-and-mouth disease poison strain in the preparation vaccine also belongs to protection scope of the present invention.
In the above-mentioned application, described vaccine is an aftosa vaccine;
In the above-mentioned application, the adjuvant that uses is IS201; The name of product of adjuvant IS201 is Montanide ISA 201VG, and available from French SEPPIC company, catalog number is L01408.
In the above-mentioned application, described foot and mouth disease is the foot and mouth disease that O type foot and mouth disease virus causes;
In the above-mentioned application, described O type foot and mouth disease virus is the pig source;
In the above-mentioned application, described O type foot and mouth disease virus is Chinese pedigree or Pan Asia pedigree or Burma's 98 pedigrees;
In the above-mentioned application, described Chinese pedigree virus stain is O/TL/Taiwan/97, and described Pan Asia pedigree virus stain is O/China/99, and described Burma 98 pedigree virus stains are O/GS/2010.
Last purpose of the present invention provides a kind of method of expanding the foot-and-mouth disease virus antigen spectrum.
The method of expansion foot-and-mouth disease virus antigen provided by the present invention spectrum comprises the steps: 1) obtain to set out foot-and-mouth disease virus genome RNA complementary cDNA; 2) coding among the described cDNA is set out the encoding gene of foot and mouth disease virus structural protein suddenlys change or the set out encoding gene of antigenic determinant of foot and mouth disease virus of the coding among the described cDNA is suddenlyd change, make the encoding gene after the sudden change identical, obtain spectrotype extensively in the genome cDNA of the purpose foot and mouth disease virus of the described foot and mouth disease virus that sets out with the allelotrope for the treatment of the foot-and-mouth disease virus resistant strain; 3) expression obtains step 2) described purpose foot and mouth disease virus.
In the aforesaid method, the described foot and mouth disease virus that sets out is the wild-type virus of arbitrary serotype, or by operating the foot and mouth disease virus that obtains arbitrarily;
In the aforesaid method, described being operating as arbitrarily cultivated or genetic manipulation;
In the aforesaid method, the described foot and mouth disease virus that sets out is preferably the wild-type virus that immunogenicity is good, replication performance is strong and virus titer is high;
In the aforesaid method, described antigenic determinant is B cell antigen determinant or T cell antigen determinant.
The foot and mouth disease virus that is obtained by above-mentioned arbitrary described method also belongs to protection scope of the present invention.
The method of expansion foot-and-mouth disease virus antigen spectrum provided by the present invention is as follows:
1, obtains the full geneome RNA complementary of strain foot and mouth disease virus cDNA fragment;
2, utilize some bases or the codon of gene manipulation techniques sudden change cDNA, make it identical with the allelotrope of epidemic isolates;
3, press foot-and-mouth disease virus gene group order, polyphone suddenlys change and the cDNA fragment of not suddenling change, and connects into the T7 promotor downstream of a plasmid vector, is built into foot-and-mouth disease virus gene group full-length infectious CDNA recombinant plasmid;
4, endonuclease linearize recombinant plasmid dna, in-vitro transcription are RNA, the transfection cultured cells; Or the recombinant plasmid dna of linearize and the recombinant plasmid dna of expressing t7 rna polymerase transfection simultaneously cultured cells; Or the clone of the expression t7 rna polymerase of the recombinant plasmid dna direct transfection of linearize cultivation, producing foot and mouth disease virus, the virus that rescue obtains is the foot and mouth disease virus of having expanded spectrotype.
In the described method 1, described foot and mouth disease virus is the wild-type virus of isolating arbitrary serotype, or by operating the foot and mouth disease virus that obtains arbitrarily, the virus that obtains with genetic manipulation that comprises cultivation, it is good to be preferably immunogenicity, and replication performance is strong, the wild-type virus that virus titer is high.Described complementary cDNA is a foot-and-mouth disease virus genome RNA through the product of reverse transcription or according to the cDNA of the artificial chemosynthesis of foot-and-mouth disease virus genome RNA sequence.
In the described method 2, described cDNA is the cDNA of the same functional area of different foot and mouth disease virus pnca gene group, being preferably each structural protein gene cDNA, more preferably is the encoding gene cDNA of antigen site, comprises B cell and T cell antigen site encoding gene cDNA.Describedly sport any technology that genetic manipulation can realize that base or codon are replaced, comprise artificial chemical synthesising technology.
In the described method 3, the cDNA of described sudden change is method 2 gained.Affiliated foot-and-mouth disease virus gene group full-length infectious CDNA comprises the T7 promotor for the cDNA fragment of sudden change and not sudden change is connected product.
In the described method 4, described recombinant plasmid dna is method 3 gained.Described cell is the clone of the foot and mouth disease virus sensitivity of vitro culture, as BHK21, IB-RS-2 and PK15 etc.; Described T7 RNA polymerase cell is the clone of carrying the T7 rna polymerase gene, is preferably the BHK21 and the IB-RS-2 clone of carrying the T7 pol gene.
The present inventor is multispectral at present south east asia popular to be O type foot and mouth disease virus, utilize the reverse genetic manipulation technology, after foot and mouth disease virus (OZK/93-08) the genome full-length cDNA sudden change strong to immunogenicity, that replication performance good (virus titer height) was modified, rescue had obtained genetically engineered virus.Several amino acid mutations are that O/TL/Taiwan/97 virus (belongs to CATHAY in this viral antigen site, the sinotype pedigree, pig source poison), several amino acid sports O/China/99 virus (genus Pan-Asia in addition, the Pan Asia pedigree) and O/GS/2010 virus (belong to SEA-Mya98, southeast hypotype Burma 98 pedigrees).With this virus as vaccine strain; propagation back deactivation on the BHK-21 cell of cultivating; make vaccine with ISA201 adjuvant (French SEPPIC) emulsification; the intramuscular inoculation immune swine; can effectively protect the attack of the capable poison of Chinese pedigree pig source and course, the capable poison of Pan Asia pedigree pig source and course and the capable poison of Burma's 98 pedigree pig sources and courses; improved the protection ratio to the capable poison of Chinese pedigree pig source and course, immune efficacy increases, and shows that spectrotype becomes wide.Therefore, utilize the reverse genetic manipulation technology can expand the spectrotype of foot and mouth disease virus.
Experimental results show that; vaccine is made in the mutated viruses strain that the present invention obtains; can resist the infringement of the capable poison of Chinese pedigree pig source and course O/TL/Taiwan/97, Pan Asia pedigree pig source and course capable poison O/China/99 and the capable poison of Burma's 98 pedigree pig sources and courses O/GS/2010 simultaneously; has the wide characteristic of spectrotype; immune swine; can obviously improve the protection ratio that the discrepant homotype foot and mouth disease virus of antigenicity is attacked, reach the immune effect of cross protection, in prevention and control foot and mouth disease, will play a significant role.
Description of drawings
Fig. 1 is the construction strategy of FMDV OZK/93-08 strain full-length cDNA molecular cloning.
Fig. 2 is the building process synoptic diagram of the foot and mouth disease virus OZK/93-08 strain complete genome infectious cDNA cloning of sudden change modification.
Fig. 3 is a mutated viruses rV-OSYNa amino acid mutation synoptic diagram.
Fig. 4 contains the 1674bp fragment of mutating acid and the enzyme of recombinant plasmid pOZK-Z123DKV is cut the evaluation electrophorogram.
Fig. 5 is A, B and the segmental electrophorogram of C.
Fig. 6 identifies electrophorogram for the reorganization plasmid enzyme restriction.
Fig. 7 infects the CPE that causes behind the BHK-21 cell for viral rV-OSyNa.
Fig. 8 is the qualification result of mutated viruses.A saves viral part of V P3, VP1 order-checking peak figure for the OZK male parent; B is modification virus rV-OSyNa part of V P3, the VP1 peak figure that checks order; C is that modification virus rV-OSyNa and OZK male parent are saved viral part of V P1 order-checking peak figure, a left side: the rV-OSyNa VP1 peak figure that checks order; Right: OZK male parent rescue virus VP 1 order-checking peak figure.
Fig. 9 detects rescue virus duplicating on BHK for indirect immunofluorescence.
Figure 10 is a rV-OSyNa rescue virus particle under the Electronic Speculum.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The public can obtain following virus strain from country of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences foot and mouth disease reference laboratory: foot and mouth disease virus OZK/93-08 strain, the capable poison of O type China pedigree pig source and course O/TL/Taiwan/97, the capable poison of O type Pan Asia pedigree pig source and course O/China/99, the capable poison of O type Burma 98 pedigree pig sources and courses O/GS/2010.Above-mentioned bacterial strains is in the preservation of the designated state man of animal doctor office of Ministry of Agriculture foot and mouth disease reference laboratory, and the public can obtain by the trust letter of animal doctor office of Ministry of Agriculture written instructions.
The capable poison of O type China pedigree pig source and course O/TL/Taiwan/97 is at document [Tsai, C.P., Pan, C.H., Liu, M.Y., Lin, Y.L., Chen, C.M., Huang, T.S., Cheng, I.C., Jong, M.H.and Yang, P.C.2000.Molecular epidemiological studies on foot-and-mouth disease type O Taiwan viruses from the 1997 epidemic.Vet.Microbiol.74 (3), 207-216.] in disclosed, provide by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.The sequence number of the genebank of this strain is: Accession No.AF030259.
The preparation and the functional verification of embodiment 1, sudden change strain
One, the full-length cDNA construction of recombinant plasmid of foot-and-mouth disease virus genome RNA correspondence
The synthetic following Oligonucleolide primers of design (Shanghai Sani company limited is synthetic):
Z1：ag
actagt 
ttgaaagggggcgctagggt
Z1-2：tggttggggggggggggggggtgaa
Z2：ttcaccccccccccccccccaacca
Z2-2：agcgtggagtcgagcacagtac
Z3：ggtctaagcaggtttccacaactg
Z3-2：aactgcagtgcttcgtgctgccctttctcaatg
Z4：gctaagcttggaactccacgaaaaggtgtcga
Z4-2:
Ttttttttttttttttttttt[annotates: restriction enzyme site and the protection base of the Nucleotide of italic for adding in addition, the back be Nucleotide in the sequence].
T1：gaaaacgcctgaggccgccgcacactgcatt
T1-2：aatgcagtgtgcggcggcctcaggcgttttc
T2：gtgaagaagatatctgactcgctctccagt
T2-2：actggagagcgagtcagatatcttcttcac
Extract total RNA of foot and mouth disease virus OZK/93-08 strain with RNeasy mini Kit (Qiagen), use Z2-2, Z3-2 and Z4-2 primer respectively, the synthetic first chain cDNA.With first chain CDNA is template, and with 4 couples of primer Z1/Z1-2, Z2/Z2-2, Z3/Z3-2 and Z4/Z4-2, amplification obtains 4 the double-stranded fragments of overlapped cDNA, called after Z1cDNA, Z2cDNA, Z3cDNA and Z4cDNA respectively.
Z3cDNA and pBluescriptSKhdv carrier are connected after using XbaI and BglII (TakaRa) digestion respectively, obtain recombinant plasmid pSK-Z3.The pBluescriptSKhdv carrier disclosed in document " " North China agronomy newspaper " 2010 the 25th the 3rd phases of volume; Cao Weijun, Li Pinghua promotes culture etc. in vain; the rescue of foot and mouth disease virus O/HN/93 vaccine strain and virus activity are identified ", and the public can obtain from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.
Utilize QuikChange@Lightning Site-Directed Mutagenesis Kit (Stratagene) rite-directed mutagenesis test kit, T1/T1-2 and T2/T2-2 are carried out rite-directed mutagenesis introducing molecular label with primer.Suddenlyd change successively respectively 2985 NotI (TakaRa) site and 4238 BglII (TakaRa) site among the recombinant plasmid pSK-Z3 obtain positive recombinant plasmid pOZK-Z3.
Template is made in Z1cDNA and Z2cDNA mixing, merges PCR with a pair of primer Z1/Z2-2, gets Z12cDNA.Z12cDNA is connected with pMD20-T plasmid (TakaRa), obtains recombinant plasmid pMD-Z12.
After pMD-Z12 and pBluescriptSKhdv plasmid vector were used the digestion of KpnI/XbaI (TakaRa) double digestion respectively, purifying reclaimed the corresponding target fragment, connected to obtain recombinant plasmid pOZK-Z12.With BglII/XbaI (TakaRa) difference double digestion digestion recombinant plasmid pOZK-Z12 and pOZK-Z3, purifying reclaims the corresponding target fragment, connects to obtain recombinant plasmid pOZK-Z123.Use BglII/NotI (TakaRa) to digest recombinant plasmid pOZK-Z123 and Z4 fragment respectively at last, purifying reclaims, and connects the recombinant plasmid pOZKF-Z1234 that obtains containing the full genome C dna clone of foot and mouth disease virus OZK/93-08 strain.Sequence verification, result have been inserted nucleotide sequence shown in the SEQ ID NO:1 between the Kpn of pBluescriptSKhdv plasmid vector I/NotI (TakaRa) site (be the DNA of the geneome RNA correspondence of virus O ZK/93-08, Z1234).
The nucleotides sequence of nucleotide sequence: Z1 is classified 1-389 position Nucleotide among the SEQ ID NO:1 as; The nucleotides sequence of Z2 is classified 365-696 position Nucleotide among the SEQ ID NO:1 as; The nucleotides sequence of Z3 is classified 588-5394 position Nucleotide among the SEQ ID NO:1 as; The nucleotides sequence of Z4 is classified 5289-8137 position Nucleotide among the SEQ ID NO:1 as.
The nucleotides sequence of the encoding gene of structural protein VP4-VP2-VP3-VP1 is classified 1660-3867 position Nucleotide among the SEQ ID NO:1 as.The encoding sequence of VP4 is a 1660-1914 position Nucleotide among the SEQ ID NO:1; The encoding sequence of VP2 is a 1915-2568 position Nucleotide among the SEQ ID NO:1; The encoding sequence of VP3 is a 2569-3228 position Nucleotide among the SEQ ID NO:1; The encoding sequence of VP1 is a 3229-3867 position Nucleotide among the SEQ ID NO:1.
Above-mentioned building process as shown in Figure 1.In the viral genome that obtains at last, 1 expression, 5 ' UTR, 2 expression L, 3 expression VP4,4 expression VP2,5 expression VP3,6 expression VP1,7 expression 2A, 8 expression 2B, 9 expression 2C, 10 expression 3A, 11 expression 3B, 12 expression 3C, 13 expression 3D, 14 expressions, 3 ' UTR.VP4, VP2, VP3, VP1 are structural protein, and L, 2A, 2B, 2C, 3A, 3B, 3C, 3D are Nonstructural Protein.
Two, sudden change modifying factor
Carrier pUC57 is available from Nanjing Genscript Biotechnology Co., Ltd., and catalog number is SD1176.
Synthetic virus strain OZK/93-08 coding region DNA (being 1056-8024 position Nucleotide among the SEQ ID NO:3), contain the sudden change of 43 of 58 of VP3, VP1 and 48 amino acids (58E → D, 43T → K and 48I → V), the synthetic DNA fragment is connected with carrier pUC57, sequence verification, obtain positive recombinant plasmid, note is made pHC.
Use following primer, make up the genome full-length cDNA recombinant plasmid pOZKF-TmFull of sudden change foot and mouth disease virus, building process as shown in Figure 2.
EapaI(+)：CC
GGGCCCAATACCTCTGGACTGGAAAC；
EsacI(-)：GTTGAAGGAGGTGGGCA
GAGCTCTCTC；
F207(+)：TACGGTGACACTAGCACTAACAACGTGCGGGGAGACCTGCAGGTGCTG；
R1629(-)：CAGCACCTGCAGGTCTCCCCGCACGTTGTTAGTGCTAGTGTCACCGTA；
EXma1(-)：TGCTTGTGTCTAGCGTCACTCG；
Exma2(-)：CTGTTTTGCGGGTGCCACGATC；
Eky2027(+)：GGTGACGTACGGGTATGCAACAGC。
With the pHC plasmid is template, with Eapal (+)/EsacI (-) primer, pcr amplification contains 58 of VP3, the purpose fragment (being 2052-3719 among the SEQ ID NO:3) of the 1674bp of 43 of VP1 and the sudden change of 48 amino acids, this fragment is through ApaI and Sac I double digestion, reclaiming the back is connected with the big fragment that the SacI double digestion reclaims through Apa I with plasmid pOZK-Z123, connect product and cut evaluation through enzyme, positive recombinant plasmid note is made pOZK-Z123DKV, enzyme is cut qualification result such as Fig. 4 (1: contain 58 of VP3, the PCR fragment of VP1 43 and the sudden change of 48 amino acids; 2:500-12000bpwide range DNA mark (500,1000,1500,2000,2500,3000,4000,5000,6000,8000,12000bp) 3: recombinant plasmid pOZK-Z123DKV Apa I and SacI enzyme are cut evaluation).
With plasmid pOZK-Z123DKV is template, use Eky2027 (+)/R1629 (-) and F207 (+)/Exma2 (-) primer right respectively, pcr amplification gets A fragment (the segmental nucleotides sequence of A is classified 2007-3681 position Nucleotide among the SEQ ID NO:3 as) and B fragment (the segmental nucleotides sequence of B is classified 3634-3836 position Nucleotide among the SEQ ID NO:3 as).The A fragment of purifying and B fragment balanced mix be as template, with EapaI (+)/Exma1 (-) be primer PCR increase C fragment (the segmental nucleotides sequence of C is classified 2052-3836 position Nucleotide among the SEQ ID NO:2 as).This C fragment contains VP1137 (137S → G), 139 (139A → T), 140 (140R → S), 141 (141V → T) and 142 (142S → N) amino acids sudden change.A fragment, B fragment, the segmental electrophorogram of C be (1:A fragment as shown in Figure 5; The 2:B fragment; 3,5:DL, 2000 (100,250,500,750,1000,2000bp); 6: merge the C fragment).
The Nucleotide that comprises the SacI site is in the upstream of design EXma1 (-) primer, so even be not with this site in the primer, also comprise in the amplified fragments.
The C fragment is inserted among the plasmid pOZK-Z123DKV that uses ApaI/SacI (TakaRa) enzymic digestion with SacI/ApaI (TakaRa) restriction endonuclease digestion back, carry out enzyme with Kpn I (TakaRa) and cut evaluation, positive recombinant plasmid note is made pOZKF-Z123m, and enzyme is cut qualification result such as Fig. 6 (1:wide range DNA mark; 2: recombinant plasmid pOZKF-TmFull XhoI enzyme is cut evaluation; 3: recombinant plasmid pOZKF-Z123m plasmid Kpn I enzyme is cut evaluation).(the C fragment has been replaced containing on the carrier pOZK-Z123DKV " 58 of VP3; about 1674bp fragment of 43 of VP1 and 48 amino acids sudden change; but this fragment still contains 58 of VP3; 43 of VP1 and 48 amino acids are suddenlyd change, but what a difference was arranged is to have contained VP1 137 (1378 → G), 139 (139A → T), 140 (140R → S), 141 (141V → T) and 142 (sudden changes of 1428 → N) amino acids) in the C fragment simultaneously
With BglII and SpeI (TakaRa) double digestion recombinant plasmid pOZKF-Z123m, reclaim big fragment; With SpeI and BglII double digestion plasmid pOZKF-Z1234, reclaim the Z4 fragment; Two fragments are connected, carry out enzyme with the XhoI restriction endonuclease and cut evaluation, qualification result as shown in Figure 6, positive recombinant plasmid note is made pOZKF-TmFull, be the foot and mouth disease virus OZK/93-08 strain complete genome infectious cDNA cloning that sudden change is modified, contain the DNA (being SEQ ID NO:2) of the full gene group RNA correspondence of mutated viruses among this plasmid pOZKF-TmFull.
The nucleotides sequence of the nucleotide sequence of mutated viruses: VP4 is classified 1660-1914 position Nucleotide among the SEQ ID NO:2 as; The nucleotides sequence of VP2 is classified 1915-2568 position Nucleotide among the SEQ ID NO:2 as; The nucleotides sequence of VP3 is classified 2569-3228 position Nucleotide among the SEQ ID NO:2 as; The nucleotides sequence of VP1 is classified 3229-3867 position Nucleotide among the SEQ ID NO:2 as.Compare with the nucleotide sequence of OZK/93-08,2740-2742 position Nucleotide is sported gat by gaa, 3355-3357 position Nucleotide sports aag by aca, 3370-3372 position Nucleotide sports gtg by atc, 3637-3639 position Nucleotide sports ggt by agt, and 3643-3654 position Nucleotide sports act agc act aac by gcc cgc gtg agc.
The aminoacid sequence of mutated viruses is shown in SEQ ID NO:4: the aminoacid sequence of VP4 is a 1-85 amino acids among the SEQ ID NO:4; The aminoacid sequence of VP2 is a 86-303 amino acids among the SEQ ID NO:4; The aminoacid sequence of VP3 is a 304-523 amino acids among the SEQ ID NO:4; The aminoacid sequence of VP1 is a 524-736 amino acids among the SEQ ID NO:4.The structural protein of mutated viruses are compared with the structural protein of OZK/93-08, and the amino acid mutation situation as shown in Figure 3.
Three, the genetically engineered virus of spectrotype has been expanded in rescue
The BHK-21 cell is supervised institute available from Chinese animal doctor, and catalog number is BHK21F5620071213.
With QIAGEN Plasmid Midi Kits (QIAGEN company) preparation plasmid pOZKF-TmFull and pcDNAT7P." " Scientia Agricultura Sinica " 2009 the 42nd the 2nd phases of volume; Li Pinghua are promoted culture Lu Cengjun etc.; disclosed in " transcribing rescue Asial type foot and mouth disease virus in the cell ", the public can obtain from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences to the pcDNAT7P plasmid in vain at document.
Behind Not I linearize pOZKF-TmFull, add Proteinase K (final concentration is 0.25 μ g/mL, Invitrogen company), behind 37 ℃ of incubation 30min, behind the purifying as transfection cDNA template.The conventional individual layer BHK-21 cell of cultivating grows at 70%～80% o'clock and is used for transfection.Respectively get 2 μ g linearize cDNA template and pcDNAT7P Lipofectamine during transfection
TMThe mediation transfection BHK-21 of 2000 (Invitrogen companies) cell.6h inhales and removes cell conditioned medium after the transfection, adding 2mL contains the DMEM substratum (Invitrogen company) of 8% foetal calf serum, puts 37 ℃ of 5%CO2 incubators, results virus behind the 72h, after freeze thawing 2-3 time, 2-3 goes down to posterity continuously on BHK-21, typical cells pathology (CPE) (Fig. 7, A: normal BHK-21 cell occur, B: the cell that CPE occurs), collect viral liquid, be purpose virus, called after rV-OSyNa.Go down to posterity on the BHK-21 cell of cultivating 30 generations of propagation with this virus ,-70 ℃ to preserve each virus standby in generation.
Four, the evaluation of viral rV-OSyNa
1, order-checking identifying virus gene
Get the continuous biography rV-OSyNa cell toxicant in 20 generations, extract the total RNA of cell toxicant with RNAasy Mini Kit, the amplification of RT-PCR method contains the specific fragment of mutating acid, and the result as shown in Figure 8.
The RT-PCR primer is:
EXma1(-)：TGCTTGTGTCTAGCGTCACTCG
Eky2599(+)：GCATCTTCCCTGTGGCTTGCTC
Eky3427(+)：CGCACCGCTACATACTACTT
The result shows: the virus genomic 2740-2742 of OZK/93-08,3355-3357, the 3370-3372 sequencing result is respectively GAA, ACA, ATC, coding E, T, three amino acid of I (Fig. 8 A), the 2740-2742 of the viral rV-OSyNa of artificial rescue, 3355-3357, the 3370-3372 sequencing result is respectively GAT, AAG, GTG, encoding D, K, three amino acid of V (Fig. 8 B), 3637-3639 and the 3643-3654 sequencing result of the viral rV-OSyNa of artificial rescue are GGT and ACTAGCACTAAC, G and T encode respectively, S, T, N amino acid (Fig. 8 C left side), and virus genomic 3637-3639 of OZK/93-08 and 3643-3654 sequencing result are AGT and GCCCGCGTGAGC, S and A encode respectively, R, V, S amino acid (Fig. 8 C right side).Show that the rV-OSyNa genome sequence that the present invention makes up is correct.
2, indirect immunofluorescence identifying virus albumen
The BHK-21 of virus inoculation (monolayer cell grows to 70%), the laggard immunofluorescence technique that in the ranks connects of 24h is identified.One anti-is O type foot and mouth disease virus rabbit positive serum, and two is anti-for adding FITC goat anti-rabbit igg (Sigma company), establishes normal cell simultaneously and contrast.Visible green specificity fluorescent in the bhk cell of virus inoculation, and the no visible fluorescence of normal cell contrast (Fig. 9, A: the BHK-21 cell of inoculation rV-OSyNa virus; B: the BHK-21 cell of virus inoculation not).Show that virus can mutually combine with O type foot and mouth disease virus rabbit positive serum.
3, electron microscopic examination
The cell toxicant 200mL of propagation back results in the BHK-21 cell after freeze thawing 2-3 time, adds the BEI deactivation, and 4 ℃ of centrifugal 40min of 6000rpm collect viral supernatant, and 4 ℃ of centrifugal 3h of 45000rpm precipitate that to add 500 μ L NET damping fluids resuspended, electron microscopic observation after the negative staining.The result is checked through diameter and is about 25nm, spheric foot and mouth disease virus particle (Figure 10).
The genetic stability analysis of 4, rescue virus
By 10% inoculum size inoculation BHK-21 cell, the time that typical cells pathology (CPE) occur is observed in continuous passage with rV-OSyNa virus, and to 9,13,30 generation virus carry out RT-PCR and detect the amino acid whose variation of rescue virus mutation.
The primer that RT-PCR detects is:
EXma1(-)：TGCTTGTGTCTAGCGTCACTCG
Eky2599(+)：GCATCTTCCCTGTGGCTTGCTC
Eky3427(+)：CGCACCGCTACATACTACTT。
3 repetitions are established in experiment.
The result: after saving viral rV-OSyNa and reaching for the 8th generation continuously with little square vase, the time of the CPE of appearance tends towards stability, about 9-10h of 100% pathology time, and little square vase 20 generation virus changes big rolling bottle over to, and 100% pathology time was about 11-12 hour, and the pathology time is stable.The sequencing result of rescue virus 9,13,30 generation VP3, VP1 gene shows do not change the amino acid stable existence of sudden change in the process of going down to posterity.
Five, vaccine production technology
A large amount of multiplication culture of 1, rescue virus
Static or rotating and culturing BHK21 cell to monolayer cell covers with, and to wherein adding viral liquid and nutrient solution, wherein viral liquid is pressed the 5-15% volume ratio and added, and adds the 1/20-1/30 of nutrient solution to the culture vessel volume; 37 ℃ are continued to cultivate, and (cytopathogenic effct CPE), gathers in the crops viral liquid and is viral cultures cytopathic effect to be occurred, and puts-20-40 ℃ preservation.
Consisting of of nutrient solution: lactoalbumin hydrolysate liquid (93-97%), amino acid solution (2-5%), microbiotic liquid (1%), HEPES liquid (0.5%) add 7.5% NaHCO
3Transfer Ph to 7.5-7.7; Described percentage composition is that volumn concentration lactoalbumin hydrolysate liquid, amino acid solution, microbiotic liquid and HEPES liquid are all available from Invitrogen company.
2, inactivation of virus
Viral liquid with a large amount of propagation of BEI (Sigma company) deactivation.
3, vaccine preparation
IS201 adjuvant products name is called Montanide ISA 201 VG, and available from French SEPPIC company, catalog number is L01408.
With inactivation of viruses liquid (antigen) and adjuvant preparation vaccine, as follows: (1) " antigen/adjuvant/antigen " two-phase adjuvant type.Inactivation of viruses liquid and IS201 adjuvant (French SEPPIC) mixing and emulsifying.The volume ratio of inactivation of viruses liquid and IS201 adjuvant is 50%: 50%, after vibration or mulser stir.Put 4-8 ℃.
The vaccine note that obtains is made the rV-O/STN vaccine.
Six, vaccine immunity animal effect comparison
48 pigs of vaccine immunity (the foot-and-mouth disease antibody titre that requires the immune pig of quilt was less than 1: 6, and it is negative to infect antibody) that step 5 prepares are measured immune efficacy.Vaccine dose 2ml/ head, immunization route are intramuscular inoculation.
Vaccine with the OZK/93-08 preparation is contrast, inoculates 48 pigs.Vaccine dose 2ml/ head, immunization route are intramuscular inoculation.The preparation method of conventional vaccine is as follows: with OZK/93-08 strain virus liquid with after the BEI deactivation, with adjuvant with 1: 1 mixed, by the foot and mouth disease inactivated vaccine rules preparation of veterinary biologics in " People's Republic of China's veterinary drug allusion quotation ".
Attack malicious method and as a result decision method all according to described in " Manual of diagnostic tests and vaccines for terrestrial animals " (version in 2009, OIE (OIE)).Specific as follows:
All immune swines behind the 28d-39d, carry out random packet by 16 every group.
The capable poison group of sinotype pig source and course: with sinotype O/TL/Taiwan/97 is that the capable poison of pig source and course carries out challenge test.
The capable poison group of Pan Asia type pig source and course: with Pan Asia type O/China/99 is that the capable poison of pig source and course is attacked the real test of poison.
The capable poison group of southeast hypotype Burma 98 pedigree pig sources and courses: carry out challenge test with the southeast hypotype Burma capable poison strain of 98 pedigree O/GS/2010 pig sources and courses.
More than every kind of virus establish 5 non-immune swines contrasts.
Attack every pig inoculation 1000ID
50Virus quantity, observe 10d, judge the immune efficacy result.Any bubble illness not occurring with the mouth hoof is protection.
Vaccination and immune efficacy data are as shown in table 1.
Table 1, vaccine immunity pig efficacy determinations result (protection number/immune number)
The result shows: the foot and mouth disease virus of modifying with suddenling change is as vaccine strain; propagation back deactivation on the BHK-21 cell of cultivating; make vaccine with ISA201 adjuvant (French SEPPIC) emulsification; immune swine can effectively be protected the attack of the capable poison of Chinese pedigree pig source and course, the capable poison of Pan Asia pedigree pig source and course and the capable poison of Burma's 98 pedigree pig sources and courses.Foot and mouth disease virus vaccine strain warp is at the amino acid whose replacement of epitope, and the vaccine immunity pig of preparation has been improved the protection ratio to the capable poison of Chinese pedigree pig source and course, and immune efficacy increases, and shows that spectrotype becomes wide.Therefore, utilize the reverse genetic manipulation technology can expand the spectrotype of foot and mouth disease virus.
Claims (10)
1. a strain hoof-and-mouth disease poison strain, the aminoacid sequence of its VP3 and VP1 structural protein is shown in 304-736 amino acids residue among the SEQ ID NO:4.
2. hoof-and-mouth disease poison strain according to claim 1 is characterized in that: the structural protein of described hoof-and-mouth disease poison strain are shown in 1-736 amino acids residue among the SEQ ID NO:4;
The encoding gene of described VP3 and VP1 structural protein is shown in 2569-3867 position Nucleotide among the SEQ ID NO:2;
The encoding gene of the structural protein of described hoof-and-mouth disease poison strain is shown in 1660-3867 position Nucleotide among the SEQ ID NO:2.
3. hoof-and-mouth disease poison strain according to claim 1 and 2 is characterized in that: the cDNA sequence of the geneome RNA correspondence of described hoof-and-mouth disease poison strain is shown in SEQ ID NO:2.
4. vaccine, its activeconstituents are arbitrary described hoof-and-mouth disease poison strain among the claim 1-3 of deactivation.
5. the application of arbitrary described hoof-and-mouth disease poison strain in the preparation vaccine among the claim 1-3.
6. vaccine according to claim 4 or the described application of claim 5 is characterized in that: described vaccine is an aftosa vaccine;
The adjuvant that uses in the described vaccine is IS201.
7. according to claim 4 or 6 described vaccines or claim 5 or 6 described application, it is characterized in that: described foot and mouth disease is the foot and mouth disease that O type foot and mouth disease virus causes;
And/or described O type foot and mouth disease virus is the pig source; And/or described O type foot and mouth disease virus is Chinese pedigree or Pan Asia pedigree or Burma's 98 pedigrees;
And/or described Chinese pedigree virus stain is O/TL/Taiwan/97, and described Pan Asia pedigree virus stain is O/China/99, and described Burma 98 pedigree virus stains are O/GS/2010.
8. a method of expanding foot-and-mouth disease virus antigen spectrum comprises the steps: 1) obtain to set out foot-and-mouth disease virus genome RNA complementary cDNA; 2) coding among the described cDNA is set out the encoding gene of foot and mouth disease virus structural protein suddenlys change or the set out encoding gene of antigenic determinant of foot and mouth disease virus of the coding among the described cDNA is suddenlyd change, make the encoding gene after the sudden change identical, obtain spectrotype extensively in the genome cDNA of the purpose foot and mouth disease virus of the described foot and mouth disease virus that sets out with the allelotrope for the treatment of the foot-and-mouth disease virus resistant strain; 3) expression obtains step 2) described purpose foot and mouth disease virus.
9. method according to claim 8 is characterized in that: the described foot and mouth disease virus that sets out is the wild-type virus of arbitrary serotype, or by operating the foot and mouth disease virus that obtains arbitrarily;
Described being operating as arbitrarily cultivated or genetic manipulation;
It is B cell antigen determinant or T cell antigen determinant that the described foot and mouth disease virus that sets out is preferably the sick described antigenic determinant of wild-type that immunogenicity is good, replication performance is strong and virus titer is high.
10. the foot and mouth disease virus that obtains by claim 8 or 9 described methods.
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