CN103074306A - Newcastle disease virus rNDV-H52AH9 and construction method and application thereof - Google Patents
Newcastle disease virus rNDV-H52AH9 and construction method and application thereof Download PDFInfo
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Abstract
The invention relates to a Newcastle disease virus rNDV-H52AH9, accession number CGMCC (China General Microbiological Culture Collection Center) No: 6654. An attenuated strain AI4 of the Newcastle disease virus is used as a carrier, HA genes of H5 and H9 subtype avian influenza virus are connected in series through FMDV (Foot and mouth disease virus) 2A and are inserted into the genome AI4 containing full-length transcript carrier pNDV/AI4 to obtain recombinant NDV genome full-length clone pNDV/AI4-H52AH9. The recombinant virus rNDV-H52AH9 is obtained by reverse genetic operation, has higher reproductive titer on embryonated eggs, and simultaneously expresses the HA protein of H5 and H9 subtype avian influenza virus stably, and lays a foundation for the development and application of multi-joint recombinant live vector vaccine of AI and ND.
Description
Technical field
The present invention relates to use Reverse Genetics, save a strain take Avian pneumo-encephalitis virus as carrier coexpression H5 and the recombinant vaccine strain rNDV-H of H9 subtype avian influenza virus hemagglutinin
52AH
9, be used for developing vaccine.
Background technology
Reverse genetic manipulation technology (Reverse genetics manipulation technique) refers to by the infective molecule cloning in the external structure RNA viruses, the virus genome RNA reverse transcription is become cDNA, on the dna molecular level, it is carried out various external manual operations, by full-length cDNA and various accessory protein assemble new RNA viruses an investigative technique [
A, Mundt E, Mebatsion T, Buchholz UJ, Mettenleiter TC.Generation of recombinant lentogenic Newcastle disease virus from cDNA.J Gen Virol 1999.80 (11): 2987-95.], also be " virus rescue ".Because the RNA viruses that final " rescue " goes out derives from the cDNA clone, therefore, can be by the artificial dna circle joint that adds in the pilot process, on dna level, the rna virus cdna group is carried out various external artificial reconstructed, solve the rna virus cdna group and be difficult to operate this difficult problem, simultaneously development and the exploitation for new generation vaccine provides new thinking [Nakaya T, et al.Recombinant Newcastle disease virus as a vaccine vector.Journal of Virology, 2001.75 (23): p.11868-73.].
Newcastle disease (ND) is one of important epidemic disease of harm aviculture, the about 15kb of Avian pneumo-encephalitis virus (NDV) genome total length, its structure is 3 '-NP-P-M-F-HN-L-5 ', the 6 kinds of structural protein of encoding successively: nucleocapsid protein (Nucleocapsid protein, NP), phosphorprotein (Phosphoprotein, P), stromatin (Matrix protein, M), fusion rotein (Fusion protein, F), hemagglutinin-neuraminic acid zymoprotein (Heamagglutinin-Neuraminidase protein, HN) and high molecular weight protein (Large protein, L).From the ND epidemic characteristic over past ten years, the NDV strain great majority that are separated to clinically belong to gene VII type virulent strain [Liu XF, Wan HQ, Ni XX, et al.Pathotypical and genotypical characterization of strain of Newcastle disease isolated from outbreaks in chicken and goose flocks in some regions of China during1985-2001.Archives of Virology, 2003,148 (7): 1387-1403.], and the genotype of conventional ND vaccine strain is mainly I and II type (such as V4, LaSota etc.), on genetic distance, differ far away with current strain isolated.
Avian influenza virus (Avian Influenza Virus, AIV) belongs to the A type Influenza Virus in the orthomyxoviridae family (Orthomyxovoridae), and viral genome is comprised of the single-stranded RNA fragment of 8 minus strands.The symptom of this virus infection bird reduces from slight upper respiratory tract infection, egg productivity, until acute whole body fatal disease.Gene element is 8 fragments, is the single-stranded RNA of negative justice, fragment 1 ~ 8 descending successively called after PB2, PB1, PA, HA, NP, NA, M and NS gene.Different demarcation hypotype according to surface antigen hemagglutinin (HA) and neuraminidase (NA); 16 kinds of HA and 9 kinds of NA hypotypes have been found so far; HA albumen is the main protection antigen of avian influenza virus, and it not only induces body to produce specific antibody, and induces ctl response.
The H5N1 subtype avian influenza virus has worldwide caused huge loss to aviculture at present, presents high incidence and high mortality, and the sound development of aviculture in serious threat, is also threatening human health simultaneously, is important infecting both domestic animals and human disease pathogen.The H9 subtype avian influenza virus perplexs the aviculture development for a long time, H9N2 is one of the popular principal mode in present Asia, extensively be present in the poultry, although performance low pathogenicity, but because propagation is extensive, can cause immunosuppression to make egg drop reduction, the sickness rate height of host Yi Fasheng secondary infection, laying hen, aviculture is caused serious harm, and had important public health meaning.
Vaccine inoculation is the main effective means of anti-these two kinds of diseases of system.Conventional vaccine has time and effort consuming, high in cost of production shortcoming.Recombinant live-vector vaccine can reach the anti-many sick effects of a pin, and saves cost, and is time saving and energy saving, is suitable for extensive immunity.The most important characteristics of vector virus are the recombinant vaccines that can be used to develop rapidly for new highly pathogenic transmissible disease.NDV is except possessing these characteristics, also have genetic stability good, do not have plurality of advantages such as possibility, high reproductive performance, the immunostimulating integrated with cellular genome are strong, can be used as vaccine carrier [Bukreyev A, Skiadopoulos M H, Murphy B R, et al.Nonsegmented negative-strand viruses as vaccine vectors[J] .Journal of Virology, 2006,80 (21): 10293-10306.].Recombinant virus in the NDV genome behind the insertion Avian Influenza Virus HA Gene; can copy in animal body and the stable HA albumen of expressing constantly; with this recombinant virus as vaccine; can induce the body generation for dual immune protective efficiency [the Veits J of AI and ND; Wiesner D; Fuchs W; et al.Newcastle disease virus expressing H5 hemagglutinin gene protects chickens against Newcastle disease and avian influenza[J] .Proc Natl Acad Sci U S A; 2006; 103 (21): 8197-8202.Schroer D; Veits J; Grund C; et al.Vaccination with Newcastle disease virus vectored vaccine protects chickens against highly pathogenic H7 avian influenza virus[J] .Avian Dis; 2009,53 (2): 190-197.]
2007; Ge Jinying etc. have reported take NDV LaSota strain as carrier; the HA gene of one strain H5N1 HPAIV is inserted the genomic P of NDV; between the M gene; recombinant virus inoculation chicken can be resisted the attack of anaphylactic type Avian pneumo-encephalitis virus and homology and allos Highly Pathogenic Avian Influenza Virus (HPAIV) simultaneously; the inoculation BALB/c mouse also can excite anti influenza protective immunity [Ge J; Deng G; Wen Z; et al.Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses[J] .Journal of Virology; 2007,81 (1): 150-158.].This recombiant vaccine on December 23rd, 2005 in Chinese official approval production, the effect of actively promoting is played in China and even world's bird flu and newcastle disease prevention and control, the development of the novel avian influenza vaccine of human is also had important reference.
But there is no at present the relevant report of inserting simultaneously two HA genes.The HA gene is typically chosen in P at the genomic on position of NDV, between the M or F, [Nakaya T between the HN gene, Cros J, Park M S, et al.Recombinant Newcastle disease virus as a vaccine vector[J] .Journal of Virology, 2001,75 (23): 11868-11873.Ramp K, Skiba M, Karger A, et al.Influence of insertion site of the avian influenza virus haemagglutinin (HA) gene within the Newcastle disease virus genome on HA expression[J] .J Gen Virol, 2011,92 (Pt2): 355-360.].The experimental data of Ramp K etc. shows that the NDV genome can hold two foreign genes that total length surpasses 3500bp simultaneously, on copying without significant adverse impact [Ramp K, Veits J, Deckers D, et al.Coexpression of Avian Influenza Virus H5 and N1 by Recombinant Newcastle Disease Virus and the Impact on Immune Response in Chickens[J] .Avian Diseases, 2011,55 (3): 413-421.].
Connection peptides can connect simultaneously a plurality of genes and guarantee polygenic coordinate expression.Foot and mouth disease virus (Foot and mouth disease virus, FMDV) 2A is from the Typical Representative of shearing polypeptide.Introducing foot and mouth disease virus between two HA genes has from the polypeptide 2A of shearing function sequence (Self-cleaving 2A peptide), two HA albumen are expressed synchronously, and property of protein does not change substantially, avoided two genes in the fusion gene owing to the obstacle in space affect certain gene activity may, one of ideal tools [the de Felipe P that makes up single vector expression polyprotein, Luke G A, Hughes L E, et al.Co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide ［ J ］ .J.Biol.Chem., 2003,278:11441-11448.].
Recklessly increased base etc. in 2011 and obtained gene VII type new castle disease virus weakening strain NDV/AI4 through Reverse Genetics, tire up to 10log
2[Hu Z, Hu S, Meng C, et al.Generation of a genotype VII Newcastle disease virus vaccine candidate with high yield in embryonated chicken eggs.Avian Dis 2011,55 (3): 391-397.].The structure [J] that contains the .clade2.3.2 H5N1 subtype avian influenza vaccine candidate strains such as eukaryon expression plasmid PHW-HA[Zhang Yan of H5 subtype avian influenza virus epidemic strain 1117 strain HA genes. journal of animal science and veterinary medicine .2012,43 (6): 915-921.] and H9N2 subtype avian influenza virus epidemic strain SD12E preserve by the Ministry of Agriculture of Yangzhou University livestock and poultry transmissible disease emphasis open laboratory.Therefore, this patent is take new castle disease virus weakening strain AI4 as carrier, and structure can be expressed the recombinant vaccine strain of H5 and H9 subtype avian influenza virus hemagglutinin simultaneously, to prevent the infection of current bird flu and newcastle disease epidemic strain.
Summary of the invention
Newcastle disease and bird flu are two large important epidemic diseases of puzzle China aviculture.The H5N1 subtype avian influenza is a kind of height fatal disease of serious harm aviculture; Although the H9 subtype avian influenza shows as low pathogenicity, sickness rate is high, often is polyinfection, causes immunosuppression, reduces laying hen and lays eggs, and causes huge financial loss to aviculture.
Therefore, it is a kind of take Avian pneumo-encephalitis virus as carrier coexpression H5 with the recombinant vaccine strain of H9 subtype avian influenza virus hemagglutinin that first purpose of the present invention is to provide, thereby solve that current gene VII type Avian pneumo-encephalitis virus, H5 and H9 subtype avian influenza virus popularity are complicated, used vaccine is many and the problem such as assorted, play the anti-many sick effects of a pin.
One strain Avian pneumo-encephalitis virus (Newcastle Disease Virus) rNDV-H
52AH
9, its preserving number CGMCC No:6654.
Described Avian pneumo-encephalitis virus rNDV-H
52AH
9Be used for simultaneous equal and express the HA albumen of H5 and H9 subtype avian influenza virus.
New castle disease virus weakening strain AI4 is gene VII type, and is consistent with the genotype of China current NDV epidemic strain; Two HA genes that insert all derive from the epidemic isolates of H5 and H9 hypotype AIV in genetic evolution.Therefore, recombinant vaccine strain rNDV-H
52AH
9Demonstrated wide application prospect in the morbidity of control current bird flu and newcastle disease, aspect popular.
It is a kind of take Avian pneumo-encephalitis virus as carrier coexpression H5 with the recombinant vaccine strain rNDV-H of H9 subtype avian influenza virus hemagglutinin that second purpose of the present invention is to provide
52AH
9Construction process: be to utilize the reverse genetic manipulation platform that causes weak goose source Avian pneumo-encephalitis virus AI4 strain of having set up, with the HA gene of the HA gene of H5N1 subtype avian influenza virus A/Chicken/Yangzhou/1117/2011 strain and the H9N2 subtype avian influenza virus A/Chicken/Shandong/12E/2010 strain 2A sequence by FMDV the be cascaded P of rear insertion transcription vector pNDV/AI4, the intergenic region of M, construct recombinant plasmid pNDV/AI4-H
52AH
9, after the transfection, obtained to have the coexpression H5 of higher reproductive performance and the recombinant vaccine strain rNDV-H of H9 subtype avian influenza virus HA albumen
52AH
9Specifically may further comprise the steps:
1) H9 hypotype AIV epidemic strain SD12E strain HA gene and NDV AI4 strain M gene fusion clone's structure
Take SD12E pnca gene group cDNA and pNDV/AI4 as template pcr amplification H9 HA gene ORF and AI4 pnca gene group 3148 ~ 3804nt and 3727 ~ 4735nt, obtain positive plasmid pH9M by Overlap PCR and molecular cloning respectively.
2) H5N1 hypotype AIV epidemic strain 1117 strain HA genes and NDV AI4 strain P gene fusion clone's structure
Respectively take the plasmid PHW-HA that contains 1117 strain HA genes and AI4 pnca gene group cDNA as template amplification HA gene (not containing terminator) and AI4 pnca gene group part P gene 2714-3141nt.Both are merged and are cloned into the pGEMT-easy carrier by molecular biology method, obtain plasmid pPH5.
3) full-length clone pNDV/AI4-H
52AH
9Acquisition
Plasmid pPH5 and pH9M carry out double digestion with Aar I and Spe I simultaneously, reclaim the purpose fragment and connect structure plasmid pPH5H9M; Then with Age I and Bstz17 I pPH5H9M is carried out the corresponding sequence of replacing pNDV/AI4 behind the double digestion, construct restructuring NDV genome full-length clone pNDV/AI4-H
52AH
9
4) recombinant virus rNDV-H
52AH
9The rescue of strain
With pCI-NP, pCI-P and three helper plasmids of pCI-L and transcription vector pNDV/AI4-H
52AH
9Cotransfection BSR-T7/5 cell, the adding final concentration is 10% SPF chick embryo allantoic liquid behind the transfection 18h, behind the 60h transfectional cell is scraped gently, fully mixes rear inoculation 9 ~ 11 age in days SPF chicken embryos with cell conditioned medium, namely obtains recombinant virus rNDV-H
52AH
9
Description of drawings
Fig. 1 recombinant plasmid pNDV/AI4-H
52AH
9Make up mode chart.
Fig. 2 recombinant plasmid pNDV/AI4-H
52AH
9EcoR I enzyme cut evaluation figure;
M：500bp?DNA?ladder?marker；1：pNDV/AI4-H
52AH
9。
Fig. 3 recombinant virus rNDV-H
52AH
9RT-PCR identify;
1,2:H5S, H5R primer extension product; M:200bp DNA ladder marker; 3,4:H9mS, H9mR primer extension product.
The detection of Fig. 4 recombinant virus infection Chinese hamster ovary celI exogenous gene expression;
A-D is the AI4 infected group, and E-H is rNDV-H
52AH
9Infected group.A, E group is take the monoclonal antibody of anti-NDV HN as primary antibodie, and B, F organizes take H5N1 hypotype AIV hyper-immune serum as primary antibodie, and C, G organize take H9N2 hypotype AIV hyper-immune serum as primary antibodie, and D, H organize and carry out indirect immunofluorescene assay take SPF chicken negative serum as primary antibodie.。
Embodiment
The coexpression H5 of the present invention take Avian pneumo-encephalitis virus as vector construction and the recombinant vaccine strain rNDV-H of H9 subtype avian influenza virus hemagglutinin
52AH
9Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, depositary institution address on October 11st, 2012: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Its preserving number CGMCC No:6654, Classification And Nomenclature is: its length of Avian pneumo-encephalitis virus (Newcastle Disease Virus) is: 18648bp.
Construction step one: thus weak Avian pneumo-encephalitis virus AI4 is the total length expressed clone's of carrier coexpression H5 and H9 subtype avian influenza virus hemagglutinin structure
Biomaterial is prepared:
Avian pneumo-encephalitis virus JS/5/05/Go strain cause weak total length expressed clone pNDV/AI4[Hu Z, Hu S, Meng C, et al.Generation of a genotype VII Newcastle disease virus vaccine candidate with high yield in embryonated chicken eggs.Avian Dis 2011,55 (3): 391-397.], the structure [J] that contains the .clade2.3.2 H5N1 subtype avian influenza vaccine candidate strains such as eukaryon expression plasmid PHW-HA[Zhang Yan of H5N1 subtype avian influenza virus epidemic strain A/Chicken/Yangzhou/1117/2011 strain HA gene. journal of animal science and veterinary medicine .2012,43 (6): 915-921.], made up by the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory, preserve; H9N2 subtype avian influenza virus A/Chicken/Shandong/12E/2010 strain is separated, is preserved by the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory.
PGEMT-easy carrier: available from Promega company.
1, the structure of fusion cloning pH9M
1) sequential analysis of H9N2 HA Gene of H 9 Subtype AIV and transgenation
Extract total RNA of H9N2 subtype avian influenza virus SD12E strain, and carry out RT-PCR with 12nt random primer (5 '-AGCAAAAGCAGG-3 ', SEQ ID NO.1) and react, obtain cDNA.Sequential analysis is found and need to be carried out silent mutation to 31 and 1063 two the Spe I restriction enzyme sites of its ORF.Design 2 pairs of mutant primers, 1 ~ 1076nt, the 1050 ~ 1683nt of the HA gene ORF that is respectively applied to increase.
Two pairs of mutant primers are respectively:
TB1S：5'ATGGAAGTAGTATCACTAATAACTATACT
CT
GTAGT?3'(SEQ?ID?NO.2)
TB1R：5'CAACCAGCAAC
AG
CCTGACCAACCTCCCTCTAT?3'(SEQ?ID?NO.3)
TB2S：5'AGGTTGGTCAGG
CT
GTTGCTGGTTGGTATGGATT?3'(SEQ?ID?NO.4)
TB2R：5'TTATATACAAATGTTGCATC?3′(SEQ?ID?NO.5)
Mutating alkali yl marks with italic.Above primer is synthetic by Shanghai biotechnology company limited.
1 ~ 1076nt zone with primer TB1S and TB1R amplification HA gene ORF obtains fragment TB1; With primer TB2S and TB2R its 1050 ~ 1683nt zone of increasing, obtain fragment TB2.
PCR reaction: be formulated as follows the PCR reaction system take the cDNA of reverse transcription as template:
PCR reaction cycle parameter: 94 ℃ of denaturation 3min, 94 ℃ of 30s, 61 ℃ of 1min ,-0.5 ℃/cycle, 72 ℃ of extensions, the extension time is 1min/kb, the rear 72 ℃ of extension 10min of 15 circulations, 4 ℃ of preservations.
With TB2R above two amplified fragments are linked to each other Overlap PCR reaction system and condition with primer TB1S by the Overlap PCR method:
Overlap PCR reaction cycle parameter: 94 ℃ of denaturation 4min; 94 ℃ of 40s, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 94 ℃ of 40s more afterwards, 62 ℃ of annealing 1min ,-0.5 ℃/cycle, 72 ℃ are extended 2min, and 10min, 4 ℃ of preservations are extended in rear 72 ℃ of 20 circulations.
Overlap PCR product links to each other with the pGEMT-easy carrier after reclaiming purifying, is converted into the bacillus coli DH 5 alpha competent cell, extracts plasmid, send Nanjing Genscript Biotechnology Co., Ltd. to carry out sequence verification behind the plasmid extraction, positive plasmid called after pmH9.
2) acquisition of pH9M
Take positive plasmid pmH9 and pNDV/AI4 as template pcr amplification H9HA gene ORF and AI4 pnca gene group 3148 ~ 3804nt, the PCR product is called after H9HA and M1 respectively respectively.
The primer sequence that is used for amplification is as follows:
H9mS：
5'TATT
ATCG
ATGGAAGTAGTATCACTAATAACTATACTGCTGGT?3'(SEQ?ID?NO.6)
H9mR：5'
GCGTGGATTTGCAAGTTATATACAAATGTTGCATCTGCAAGACC?3'(SEQ?ID?NO.7)
M1S：5'
CATTTGTATATAACTTGCAAATCCACGCTTCAACACCC?3'(SEQ?ID?NO.8)
M1R：5'TCTAGGGCTCCGCTCCCAGGG?3'(SEQ?ID?NO.9)
Restriction enzyme site marks with italic, and the 2A sequence is marked by wavy line, the following underlining of lap.Above primer is synthetic by Shanghai biotechnology company limited.
PCR reaction: be formulated as follows the PCR reaction system take pmH9 or pNDV/AI4 as template:
PCR reaction cycle parameter: 94 ℃ of denaturation 3min, 94 ℃ of 30s, 64 ℃ of annealing 1min ,-1 ℃/cycle, 72 ℃ are extended 2min30s, 94 ℃ of 40s again after 8 circulations, 58 ℃ of annealing 1min, 72 ℃ are extended 2min30s, the rear 72 ℃ of extension 10min of 15 circulations, 4 ℃ of preservations.
Above two amplified fragments H9HA are linked to each other by Overlap PCR with M1 with M1R with primer H9mS.Overlap PCR reaction system and condition:
Overlap PCR reaction cycle parameter: 94 ℃ of denaturation 3min, 94 ℃ of 30s, 64 ℃ of 1min,-0.5 ℃/cycle, 72 ℃ of extensions, the extension time is 1min/kb, the rear 94 ℃ of 45s of 16 circulations, 58 ℃ of 1min, 72 ℃ of extensions, the extension time is 1min/kb, after 8 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.
Overlap PCR product is through reclaiming the purifying rear clone to the pGEMT-easy carrier, and the plasmid of extraction send Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, positive plasmid called after pmH9M1.
In addition, again take pNDV/AI4 as template pcr amplification AI4 pnca gene group 3727 ~ 4735nt, PCR product called after MF.
The primer sequence that is used for amplification is as follows:
MFS：5'GTCGAATAGGTACTCATCAG?3″(SEQ?ID?NO.14)
MFR：5'TATGATTGACCCTGTCTGAG?3″(SEQ?ID?NO.15)
Above primer is synthetic by Shanghai biotechnology company limited.
PCR reaction: be formulated as follows the PCR reaction system take pNDV/AI4 as template:
PCR reaction cycle parameter: 94 ℃ of denaturation 3min, 94 ℃ of 30s, 61 ℃ of annealing 1min ,-0.5 ℃/cycle, 72 ℃ are extended 1min30s, after 15 circulations again 72 ℃ extend 10min, 4 ℃ of preservations.
PCR product MF is through reclaiming the purifying rear clone to the pGEMT-easy carrier, and the plasmid of extraction send Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, positive plasmid called after pMF.
Plasmid pMF and pmH9M1 carry out double digestion with Kas I and Spe I respectively, reclaim purpose fragment and connection, obtain plasmid pH9M(and see Fig. 1).
2, the structure of fusion cloning pPH5
2714-3141nt take plasmid PHW-HA and pNDV/AI4 as template amplification HA gene ORF (not containing terminator) and AI4 pnca gene group.
Two pairs of primers that are used for amplification are respectively:
PS：5'GGGCATGATGAAGATCCTGGACCCTGGTTGTG?3'(SEQ?ID?NO.10)
PR：5'
TTCTACCCGTATTTTTTCTAAGAGGAGCTTGGTGCAGATACCGTG?3'(SEQ?ID?NO.11)
H5S：5'
TTAGAAAAAATACGGGTAGAA 
ATGGAGAAAATAGTGCTTCTCTTTG?3'(SEQ?ID?NO.12)
H5R：5'TATT
ATCGTCAAAATGCAAATTCTGCACTGTAACG?3'(SEQ?ID?NO.13)
Restriction enzyme site marks with italic, and ACGGGTAGAA is NDV transcription initiation signal sequence, and TTAGAAAAAA is NDV transcription termination signal sequence, and the Kozak sequence is marked by double underline, the following underlining of lap.Above primer is synthetic by Shanghai biotechnology company limited.
PCR reaction: be formulated as follows the PCR reaction system take PHW-HA or pNDV/AI4 as template:
PCR reaction cycle parameter: 94 ℃ of denaturation 3min, 94 ℃ of 30s, 63 ℃ of 1min, 72 ℃ of extensions, the extension time is 1min/kb, the rear 72 ℃ of extension 10min of 20 circulations, 4 ℃ of preservations.
Above two amplified fragments are linked to each other by the Overlap PCR method with H5R with primer PS.Overlap PCR reaction system and condition:
Overlap PCR reaction cycle parameter: 94 ℃ of denaturation 4min; 55 ℃ of annealing 1min, 72 ℃ are extended 2min, 94 ℃ of 40s more afterwards, 63 ℃ of annealing 1min ,-0.5 ℃/cycle, 72 ℃ are extended 2min30s, and 10min, 4 ℃ of preservations are extended in rear 72 ℃ of 20 circulations.
Overlap PCR product enters the pGEMT-easy carrier through reclaiming the purifying rear clone, and the plasmid of extraction send Nanjing Genscript Biotechnology Co., Ltd. to carry out sequence verification, positive plasmid called after pPH5.
3, recombinant plasmid pNDV/AI4-H
52AH
9Acquisition
Respectively plasmid pPH5 and pH9M are carried out double digestion with Spe I and Aar I, obtain plasmid pPH5H9M through the molecular cloning operation; At last pPH5H9M is replaced the corresponding sequence of transcription vector pNDV/AI4 after Age I and Bstz17 I enzyme are cut, recombinant plasmid is cut evaluation with EcoR I enzyme, positive colony called after pNDV/AI4-H
52AH
9(seeing Fig. 1 and Fig. 2).
More than each toolenzyme available from Thermo Fisher Scientific.
Step 2: recombinant virus rNDV-H
52AH
9The rescue of strain
Biomaterial is prepared
PCI-NP, pCI-P, pCI-L eukaryon expression plasmid [Hu Shunlin, Yanmei ZHANG, Sun Qing etc. the foundation [J] of goose source Avian pneumo-encephalitis virus rescue system. microbiology circular .2007,34 (3): 426-429.]: make up, preserve by the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory.
BSR-T7/5 cell: be so kind as to give by the step researcher of CHIGO of Harbin Veterinary Medicine Inst., China Academy of Agriculture.
Gene VII type Monoclonal Antibodies To Newcastle Disease Virus 6B1[Hu Shunlin. development, evaluation and the Preliminary Applications [D] of gene VII type Monoclonal Antibodies To Newcastle Disease Virus. Yangzhou: the .2004. of Yangzhou University]: developed by the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory.
1, the rescue of recombinant virus
With 5 * 10
5The BSR-T7/5 cell is inoculated in the 35mm Tissue Culture Dish, spends the night with the DMEM culture medium culturing that contains 1mg/mL G418 and 10% calf serum, and about 60% ~ 80% is used for transfection when being paved with.After the used plasmid of transfection is all used QIAprep Spin Miniprep Kit extracting, measure concentration and purity, concentration more than 0.1 μ g/ μ l and the plasmid of purity between 1.6 ~ 1.8(OD260/280) be used for transfection.With pCI-NP, pCI-P and three eukaryon expression plasmids of pCI-L and total length transcription vector pNDV/AI4-H
52AH
9Cotransfection BSR-T7/5 cell, adding final concentration behind the 18-24h is the 10%SPF embryo allantoic liquid, behind the 60h transfection sample is sealed with sealed membrane (parafilm), transfer to-70 ℃ of cryogenic refrigerator multigelations 2 ~ 3 times, then transfectional cell is scraped gently, fully mix rear inoculation 9 ~ 11 age in days SPF chicken embryos with cell conditioned medium, 3 pieces of each sample inoculations, 0.4mL/ piece, hatch for 37 ℃.Regularly according to embryo, discard dead embryo in the 24h.Dead embryo places 4 ℃ of fully coolings after taking out 24h, collects allantoic fluid after chicken embryo vasoconstriction, namely obtains the recombinant virus rNDV-H of coexpression H5 and H9 subtype avian influenza virus hemagglutinin
52AH
9
The allantoic fluid of collecting records HA by the OIE standard and tires and be 9log
2, compare 1 titre that descended with maternal VII d hypotype strain JS/5/05/Go weakening strain NDV/AI4, and the HA characteristic can be suppressed by the monoclonal antibody 6B1 for NDV HN albumen.
2, recombinant virus rNDV-H
52AH
9RT-PCR identify
HA and HI are detected all positive allantoic fluid after SPF chicken embryo uploaded for two generations, collect allantoic fluid, extracting RNA is used for the RT-PCR reaction, utilize increase the respectively HA gene of H5 and H9 of primer H5S, H5R and H9mS, H9mR, length is about 1600bp, reclaim evaluations of checking order of purpose fragment, the HA gene of two strains of sequencing result demonstration of amplified fragments all successfully inserts (see figure 3).
Be used for amplification recombinant virus rNDV-H
52AH
9The primer sequence of middle HA gene is:
H5S：5'TTAGAAAAAATACGGGTAGAAGCCACCATGGAGAAAATAGTGCTTCTCTTTG?3'
H5R：5'TATTCACCTGCATCGTCAAAATGCAAATTCTGCACTGTAACG?3'
H9mS：
5'TATTCACCTGCATCGTTGAATTTTGACCTTCTTAAGCTTGCGGGAGACGTCGAGTCCAACCCTGGGCCCATGGAAGTAGTATCACTAATAACTATACTGCTGGT?3'
H9mR：5'GCGTGGATTTGCAAGTTATATACAAATGTTGCATCTGCAAGACC?3'
Step 3: the Identification of Biological Characteristics of recombinant virus
Biomaterial is prepared:
SPF kind egg is hatched in the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory available from Beijing Cimmeria company.
Anti-NDV HN protein-specific monoclonal antibody 6B1, H5N1 hypotype AIV hyper-immune serum, H9N2 hypotype AIV hyper-immune serum are by the preparation of the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory, preservation.
The anti-chicken IgG of the rabbit of the sheep anti-mouse igg of FITC mark, the FITC mark IgG of FITC mark (below be collectively referred to as): available from Sigma company.1, the mensuration of MDT and ICPI
With continuous 10 times (10 respectively of sterile saline
-3, 10
-410
-7) increase progressively and dilute recombinant virus rNDV-H
52AH
9, 5 piece of 9～11 age in days SPF chicken embryo of each extent of dilution inoculation hatched 5d for 37 ℃, presses the MDT that virus is calculated in the OIE standard method; The allantoic fluid of recombinant virus is pressed the ICPI that virus is calculated in the OIE standard method with the SPF chicken of 10 times of dilutions of sterile saline by 10 1 ages in days of intracranial inoculation.
A virus 5 dilution inoculated into chick embryo does not occur dead in 120h, illustrates that the MDT value of this strain is greater than 120h; Allantoic fluid is behind intracranial inoculation 1 age in days SPF chicken, and the measured value of ICPI is 0.22 only, shows that the virulence of the virus of being rescued meets the standard of low virulent strain fully.
2, rNDV-H
52AH
9The expression of foreign gene
With recombinant virus rNDV-H
52AH
9Viral AI4 does respectively 10 with female parent
-3Inoculation Chinese hamster ovary celI after the dilution, behind the 48h, respectively take anti-NDV HN protein-specific monoclonal antibody 6B1, H5N1 hypotype AIV hyper-immune serum, H9N2 hypotype AIV hyper-immune serum as primary antibodie, the IgG of FITC mark is two anti-, carries out the IFA test.All can observe specificity fluorescent signal (Fig. 4 .E, F, G) in the recombinant virus-infected cell hole; Maternal virus infected cell hole is that the primary antibodie interaction energy detects fluorescence with 6B1, and other then can not observe positive reaction (Fig. 4 .A, B, C).Recombinant virus and the IFA result of maternal virus infected cell take the SPF negative serum as primary antibodie negative (Fig. 4 .D, H).
Claims (3)
1. a strain Avian pneumo-encephalitis virus (Newcastle Disease Virus) rNDV-H
52AH
9, its preserving number CGMCC No:6654.
2. the described Avian pneumo-encephalitis virus rNDV-H of claim 1
52AH
9Be used for simultaneous equal and express the HA albumen of H5 and H9 subtype avian influenza virus.
3. the described Avian pneumo-encephalitis virus rNDV-H of claim 1
52AH
9Construction process, it is characterized in that: take the genome total length transcription vector pNDV/AI4 of VIId hypotype newcastle disease weakening strain AI4 as skeleton, express simultaneously the HA gene of the popular AIV strain of two strains H5 hypotype AIV A/Chicken/Yangzhou/1117/2011 and H9 hypotype AIV A/Chicken/Shandong/12E/2010; After soon the HA gene of H5 and H9 hypotype AIV will be cascaded by foot and mouth disease virus 2A gene, between the P of insertion transcription vector pNDV/AI4, the M gene, obtain recombinant Newcastle disease virus Genomic full_length cDNA clone pNDV/AI4-H
52AH
9Obtain recombinant virus rNDV-H behind this plasmid and the helper plasmid cotransfection BSR-T7/5 cell
52AH
9
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| CN103468651A (en) * | 2013-10-08 | 2013-12-25 | 扬州大学 | Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof |
| CN103525774A (en) * | 2013-10-08 | 2014-01-22 | 扬州大学 | Recombinant newcastle disease virus rAI4-gB vaccine strain expressing infectious laryngotracheitis virus gB protein, and construction method for the vaccine strain |
| CN107312872A (en) * | 2017-06-22 | 2017-11-03 | 广州市华南农大生物药品有限公司 | Differentiate multiple PCR primer group, kit and the detection method of H5 subtype avian influenza virus and its NA hypotypes simultaneously |
| CN107384875A (en) * | 2017-09-01 | 2017-11-24 | 扬州大学 | Chimeric the newcastle Disease poisonous carrier H7 live vaccines Candidate Strain and its construction method for overcoming chicken Newcastle disease maternal antibody to influence |
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| CN103468651A (en) * | 2013-10-08 | 2013-12-25 | 扬州大学 | Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof |
| CN103525774A (en) * | 2013-10-08 | 2014-01-22 | 扬州大学 | Recombinant newcastle disease virus rAI4-gB vaccine strain expressing infectious laryngotracheitis virus gB protein, and construction method for the vaccine strain |
| CN110505879A (en) * | 2016-12-14 | 2019-11-26 | 勃林格殷格翰动物保健美国公司 | Express the recombinant hvt carrier of a variety of antigens and application thereof of poultry pathogens |
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| CN107312872A (en) * | 2017-06-22 | 2017-11-03 | 广州市华南农大生物药品有限公司 | Differentiate multiple PCR primer group, kit and the detection method of H5 subtype avian influenza virus and its NA hypotypes simultaneously |
| CN107384875A (en) * | 2017-09-01 | 2017-11-24 | 扬州大学 | Chimeric the newcastle Disease poisonous carrier H7 live vaccines Candidate Strain and its construction method for overcoming chicken Newcastle disease maternal antibody to influence |
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