CN103468651A - Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof - Google Patents
Recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and generating method thereof Download PDFInfo
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Abstract
The invention discloses a recombination Newcastle vaccine strain rAI4-S1 for expressing infectious bronchitis virus S1 protein and a generating method of the recombination Newcastle vaccine strain rAI4-S1. The conservation number of the recombination Newcastle vaccine strain rAI4-S1 is CGMCC No: 8054. According to the generating method of the recombination Newcastle vaccine strain rAI4-S1, a built reverse genetic manipulation platform of a gene VII type NDV attenuated virus AI4 strain is utilized, the sequence revealed in the SEQ ID NO.7 is inserted into an AI4 strain genome full-length transcription carrier pNDV/AI4, and then the recombination Newcastle vaccine virus genome full-length cDNA clonal pNDV/AI4-S1 containing infectious bronchitis virus S1 genes is obtained. According to a recombination virus obtained through transfection, a chick embryo has high breeding titer, the S1 protein still can be expressed after continuous passage, and the generating method is suitable for large-scale production of vaccines and can be used for processing the vaccines.
Description
Technical field
The present invention relates to apply Reverse Genetics, save a strain and take the recombinant vaccine strain rAI4-S1 that Avian pneumo-encephalitis virus is the vector expression S 1 protein of infectious bronchitis virus, for developing vaccine.
Background technology
Reverse genetic manipulation technology (Reverse genetics manipulation technique): for RNA viruses, Reverse Genetics refers in vitro by building the infective molecule cloning of RNA viruses, the virus genome RNA reverse transcription is become to cDNA, on the DNA molecular level, it is carried out to various external manual operations, assembled an investigative technique of new RNA viruses by full-length cDNA and various accessory protein, also be the full-length infectious CDNA clones technology, often be called as again " virus rescue ".[Neumann?G,Whitt?M?A,Kawaoka?Y,et?al.(2002).A?decade?after?the?generation?of?a?negative-sense?RNA?virus?from?cloned?cDNA–what?have?we?learned?J?Gen?Virol.83(11):2635-2662.]。The Avian pneumo-encephalitis virus Reverse Genetics is to set up on the basis of the reverse genetics operative technique of the minus-stranded rna virus with reference to other sub-thread non-segmented negative (NNSV).[the Peeters BPH such as Peeters in 1999, Olays, oe Leeuw, Koch G, et al. (1999) .Rescue of Newcastle disease virus from cloned cDNA:Evidence that cleavability of the fusion protein is a major determinant for virulence.J Virol.73:5001-5009.] and [the Radecke F such as Romer-Oberdorfer, Spielhofer P, Schneider H, et al. (1995) .Rescue of meales virus from cloned DNA.EMBO J14:5773-5784.] almost simultaneously reported first the successful rescue of Avian pneumo-encephalitis virus.
Newcastle disease (Newcastle disease), claim again philippine fowl disease, pseudo-checken pest etc.Caused a kind of acute, the septic of the poultry such as main infection chicken, dove, quail, turkey and wild bird, height contagious disease by Avian pneumo-encephalitis virus.Can infect the bird more than 240 kinds, this disease is all attached great importance in countries in the world.OIE (OIE) classifies it as that animal is legal must report transmissible disease.Avian pneumo-encephalitis virus (Newcastle disease virus, NDV) is the minus-stranded rna virus of sub-thread, non-segmented negative, belongs to Paramyxoviridae, paramyxovirus subfamily, fowl Rubulavirus member.Pathogenic index according to standard can be divided into virus anaphylactic type (strong poison), middle hair style (mesogenic) and delayed type (weak poison).The about 15kb of genome length, its structure is 3 '-leader-NP-P-M-F-HN-L-trailer-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).Wherein F albumen has the effect that inducing cell merges, destroys cell, and F albumen is the weak major cause of different N DV strong toxicity, and the cracking site of F albumen has played vital role.[Peeters?BP,et?al.Rescue?of?Newcastle?disease?virus?from?cloned?cDNA:evidence?that?cleavability?of?the?fusion?protein?is?a?major?determinant?for?virulence.Journal?of?Virology,1999.73（6）:p5001-9.]。
Chicken infectious bronchitis (Infectious bronchitis, IB) be that the chicken that caused by virus is acute, the diseases such as respiratory tract of height contagious infection, it is characterized by the cough of disease chicken, sneeze, send the tracheae rale, chick, runny nose can also occur, laying hen occurs laying eggs and reduces and the rotten phenomenon of quality, cause and support the huge financial loss [Yin Zhen of fowl, Liu Jinghua. animal virology [M]. the 2nd edition. Beijing: Science Press, 1997,675-680.].S albumen plays an important role to the IBV infectivity, when virus particle starts when sensitive cells is infected, is at first that the S albumen of IBV virion cyst membrane combines with the acceptor of sensitive cells, and fusion or interior pinocytosis by virus envelope and cytolemma enter cell.
The characteristics of IBV are that variation is frequent, the serotype complexity, the clinical manifestation of associated diseases differs greatly, from (1931) reported first such as Schalk classical breathing pattern infectious bronchitis of fowl and be defined as virus disease since, the multiple pathotypes such as existing visible peristalsis visible intestinal peristalsis, Glandular Stomach Type, kidney type are reported in succession.Various places mainly adopt the method for vaccine inoculation to prevent IB at present; but because its variant occurs than very fast; each strain Virulence Difference is large; between strain, cross-protection is low; even in the situation that immunity is good; the chicken group still is subject to the threat of IBV variant, and the sound development of poultry husbandry in serious harm.
Vaccine inoculation is the main effective means of anti-newcastle disease processed and these two kinds of diseases of infectious bronchitis.But deactivation vaccine can not provide enough protections, thereby can causing local untoward reaction, oil emulsion adjuvant causes the discomfort of meat decline and animal.Attenuated live vaccine is by different means; make viral virulence attenuation of or forfeiture; body does not occur or very light clinical symptom occurs after accepting this vaccine; stimulate the immunity system of body to produce the immune response viral for this; while making it to contact afterwards this virus, protect the ill or not ill clinical course of body lighter.Can inoculate by natural way (collunarium, eye droppings, oral etc.), so not only can produce systemic immune response, and can induce and produce the local immunity reaction, only need inoculation once just can reach satisfied effect.
The most important characteristics of vector virus are to be used to develop rapidly the recombinant vaccine 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 after the insertion foreign gene, can copy in animal body and stablize and express constantly foreign protein, with this recombinant virus as vaccine, can induce body to produce dual immune protective efficiency [the Veits J for 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.], [the Lamb such as Lamb, R.A., Kolakofsky, D., et al. (2001) .Paramyxoviridae:the viruses and their replication, p.1305-1340.In D.M.Knipe and P.M.Howley, Fields virology.Lippincott Williams& Wilkins Philadephia, Pa.] study and find that transcribing of paramyxovirus is polar effect, more the closer to its transcription product of gene of 3 ' end, lower the closer to the amount of 5 ' end transcription product.From current research, when foreign gene inserts between NP gene or P and M gene, its expression amount is higher, and these two positions become the first-selection that foreign gene inserts.Therefore, chosen the S1 gene that inserts infectious bronchitis virus between P and M gene in this research.
Successfully built full-length clone pNDV/AI4 after the F protein cleavage site mutation of the VIId hypotype NDV strain JS5/05 that Hu Zenglei in 2011 etc. separate this laboratory, obtained attenuated virus AI4 through reverse genetic manipulation, the HA of its allantoic fluid tires as 10log2[Hu Z, Hu S, Meng C, et al.Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with HighYield in Embryonated Chicken Eggs.Avian Diseases2011,55 (3): 1759-1769].2006, the Ministry of Agriculture of Yangzhou University livestock and poultry transmissible disease emphasis open laboratory was separated to strain nephropathic type infectious bronchitis virus CK/CH/JS/06 II [Wu Peipei, Tang Yinghua, a Liu Wenbo etc. in 2006 from Hai'an, Jiangsu morbidity chicken group.The Identification of Biological Characteristics of infectious bronchitis virus strain isolated and Genetic Variation Analysis thereof [J]. journal of animal science and veterinary medicine .2009,40(1): 89-92.].Therefore, the present invention on the basis of the above, be take pNDV/AI4 as skeleton, and structure can be expressed the recombinant Newcastle disease vaccine strain of S 1 protein of infectious bronchitis virus, for tackling the popular of current infectious bronchitis and newcastle disease.
Summary of the invention
First purpose of the present invention is to invent a kind of recombinant vaccine strain that Avian pneumo-encephalitis virus is vector expression infectious bronchitis virus S1 gene of take, for the manufacture of vaccine.
The present invention expresses the recombinant vaccine strain rAI4-S1 of infectious bronchitis virus S1 gene, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 5th, 2013, its preserving number CGMCC No:8054.
The reverse genetic manipulation platform of the Avian pneumo-encephalitis virus AI4 strain that utilization of the present invention has been set up, between the P of the S1 gene insertion NDV total length transcription vector pNDV/AI4 of the strain infectious bronchitis virus that the Ministry of Agriculture of Yangzhou University livestock and poultry transmissible disease emphasis open laboratory is separated, M gene, construct recombinant plasmid pNDV/AI4-S1, after this recombinant plasmid and three helper plasmid cotransfection BSR-T7/5 cells, obtain the recombinant vaccine strain rAI4-S1 of the expression infectious bronchitis virus S1 gene with higher reproductive performance, be applicable to the scale operation of vaccine.
Second purpose of the present invention is to provide a kind of construction process of expressing the recombinant vaccine strain rAI4-S1 of infectious bronchitis virus S1 gene, that to take the genome total length transcription vector pNDV/AI4 that gene VII type epidemic strain NDV causes weak malicious AI4 strain be skeleton, between the P of the insertion of the sequence shown in SEQ ID NO.7 AI4 pnca gene group total length transcription vector pNDV/AI4, M gene, through Reverse Genetics, obtain recombinant virus rAI4-S1.
Sequence shown in described SEQ ID NO.7 is the S1 gene of infectious bronchitis virus Jiangsu strain isolated CK/CH/JS/06 II, GenBank accession number: EU031526.This sequence can obtain by the clone, also can directly synthesize.
Method of the present invention specifically comprises the following steps:
1. the structure of full-length clone pNDV/AI4-S1
1) clone of infectious bronchitis virus S1 gene
Extract the geneome RNA of infectious bronchitis virus by the Trizol method, experimental design primer S1-F and S1-R be take viral genome as template amplification S1 gene, clone in the pCR2.1 carrier and build pCR2.1-S1, wherein S1 gene fragment two ends are with Apa I restriction enzyme site.
2) structure of interstitial granules pUCAI4-PM in
Take pNDV/AI4 as template, by the segment area of primer AI4-F and AI4-R amplification AI4 genome 2851-4747nt, amplified fragments and pUC18 carrier are carried out to double digestion by Xba I and Hind III simultaneously, reclaim the purpose fragment and connect, build plasmid pUCAI4-PM.
3) the S1 gene clone advances in pUCAI4-PM to build pUCAI4-PM-S1
Respectively positive plasmid pCR2.1-S1 and pUCAI4-PM are cut with Apa I enzyme, reclaim the purpose fragment, the pUCAI4-PM enzyme is cut to product and spend the phosphorylation agent box and carry out connecting again after the dephosphorylation processing, construction recombination plasmid pUCAI4-PM-S1.
4) acquisition of full-length clone pNDV/AI4-S1
Plasmid pUCAI4-PM-S1 and pNDV/AI4 carry out double digestion by Age I and BstZ17 I simultaneously, reclaim the purpose fragment and connect, and construct restructuring NDV genome full-length clone pNDV/AI4-S1, and concrete construction process is shown in Fig. 1.
2. the rescue of recombinant virus pNDV/AI4-S1 strain
By total length plasmid pNDV/AI4-S1 and pCI-NP, pCI-P and tri-helper plasmid cotransfection BSR-T7/5 cells of pCI-L, inoculate 9～11 age in days SPF chicken embryos after 60h, obtain recombinant virus rAI4-S1 after a blind passage generation.
The accompanying drawing explanation
Fig. 1 recombinant plasmid pNDV/AI4-S1 builds mode chart.
The detection of Fig. 2 recombinant virus infection CEF cell exogenous gene expression..A-C be the AI4 infected group, D-F is the rAI4-S1 infected group.It is primary antibodie that A, D group be take the how anti-chicken serum of anti-NDV, and B, E group be take the IBV chicken serum and resisted as primary antibodie more, and C, F group be take SPF chicken negative serum and carried out indirect immunofluorescene assay as primary antibodie.
Embodiment
The recombinant Newcastle disease vaccine strain rAI4-S1 that the present invention expresses S 1 protein of infectious bronchitis virus is preserved in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 5th, 2013, Institute of Microorganism, Academia Sinica), Classification And Nomenclature is Avian pneumo-encephalitis virus rAI4-S1, preserving number CGMCC No:8054.
Construction step one: take the structure of Avian pneumo-encephalitis virus AI4 as the total length expressed clone of vector expression S 1 protein of infectious bronchitis virus
Biomaterial is prepared:
The total length expressed clone pNDV/AI4(Hu Z of Avian pneumo-encephalitis virus AI4 strain, Hu S, Meng C, et al.Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with HighYield in Embryonated Chicken Eggs.Avian Diseases2011,55 (3): 1759-1769) by the Ministry of Agriculture of Yangzhou University livestock and poultry transmissible disease emphasis open laboratory, build, preserve.
Infectious bronchitis virus strain (Jiangsu strain isolated CK/CH/JS/06 II) [Wu Peipei, Tang Yinghua, Liu Wenbo etc. the Identification of Biological Characteristics of infectious bronchitis virus strain isolated and Genetic Variation Analysis thereof [J]. journal of animal science and veterinary medicine .2009,40 (1): 89-92.] separate, preserve by the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory.
PCR2.1 carrier: purchased from Invitrogen company; PUC18 carrier, Dephospharylation (BAP) Kit: purchased from precious biotechnology (Dalian) company limited; AMV ThermoScript II, High fidelity DNA polymerase, T4DNA ligase enzyme, Agarose Gel DNA ExtractionKit are purchased from Roche company; Transfection reagent SuperFect and plasmid extraction test kit (QIAprep Spin MiniPrep Kit) are QIAGEN company product; All the other conventional reagent are domestic analytical pure.
1, the sequential analysis of infectious bronchitis S1 gene and transgenation
The Trizol method is extracted the full geneome RNA of infectious bronchitis virus, according to the IBVCK/CH/JS/06 II strain S1 genome sequence logged on GenBank (GenBank accession number: EU031526) analyze and find that needs first carry out silent mutation to the BstZ17 I restriction enzyme site of 1262 of its ORF.Design 2 pairs of primers, 1～1272nt, the 1248～1620nt segment area of the S1 gene ORF that is respectively used to increase.
Two pairs of mutant primers are respectively:
S1-F:5′-ATGGGCCCTTAGAAAAAATACGGGTAGAAGCCACCATGTTGGGGAAGTCACTGTTTTTAG-3′（SEQ?ID?NO.1）
S1-R1:5′-AGTGATGGCTCTCGCATACAGACTAGA-3′（SEQ?ID?NO.2）
S1-F1:5′-GTTCTAGTCTGTATGCGAGAGCCATCA-3′（SEQ?ID?NO.3）
S1-R:5 '-CTGGGCCC
tTAaCGCCTGCGACGATGTGAGCTATTG-3 ' (Apa I) (SEQ ID NO.4) mutating alkali yl marks with italic, and underscore means the base increased, and upper primer is synthetic by Shanghai biotechnology company limited.
PCR reaction: take and pass a virus genome RNA and be formulated as follows the PCR reaction system as template:
| 10×High?Fidelity?Buffer | 2.5μL |
| Sense?Primer(50μmol/L) | 0.5μL |
| Reverse?Primer(50μmol/L) | 0.5μL |
| dNTPs(10mmol/L) | 1μL |
| Expand?High?Fidelity?Polymerase(3.5U/μL) | 0.5μL |
| Virus genome RNA | 2μL |
| Mg 2+ | 0.5μL |
| SW | 17.5μL |
PCR reaction cycle parameter: 94 ℃ of denaturation 5min, 94 ℃ of 50s, 60 ℃ of 1min15s, 72 ℃ of extensions, the extension time is 1min/kb, 15 rear 72 ℃ of extension 10min of circulation, 4 ℃ of preservations.
Overlap PCR reaction system and condition:
| 10×High?Fidelity?Buffer | 2.5μL |
| Primer?S1-F(50μmol/μL) | 0.5μL |
| PrimerS1-R(50μmol/μL) | 0.5μL |
| dNTPs(10mmol/L) | 1μL |
| Expand?High?Fidelity?Polymerase(3.5U/μL) | 0.5μL |
| The S1-1 amplified fragments | 2μL |
| The S1-2 amplified fragments | 2μL |
| SW | 16μL |
PCR reaction cycle parameter: 94 ℃ of denaturation 5min; 94 ℃ of 50s, 58 ℃ of annealing 1min, 72 ℃ are extended 2min, 94 ℃ of 40s again after 2 circulations, 64 ℃ of annealing 1min, 72 ℃ are extended 2min, and 10min, 4 ℃ of preservations are extended in latter 72 ℃ of 15 circulations.
1～1272nt segment area with primer S1-F and S1-R1 amplification S1 gene ORF, by primer S1-F1 and S1-R its 1248～1620nt segment area that increases, the PCR product is labeled as respectively S1-1 and S1-2 after reclaiming purifying, with primer S1-F and S1-R, by above-mentioned two fragments, the method by Overlap PCR merges, silent mutation restriction enzyme site BstZ17 I.Reclaim the purpose fragment and be connected with the pCR2.1 carrier, be converted into the bacillus coli DH 5 alpha competent cell, extract plasmid, positive plasmid called after pCR2.1-S1.
2, the structure of plasmid pUCAI4-PM
Take pNDV/AI4 as template, with the 2851-4747nt sequence fragment in primer AI4-F and AI4-R amplification AI4 genome, fragment and pUC18 carrier are carried out to double digestion by Xba I and Hind III simultaneously, reclaim the purpose fragment and connect, be converted into the bacillus coli DH 5 alpha competent cell, extract plasmid, positive plasmid called after pUCAI4-PM.
Amplimer is as follows:
AI4-F:5′-AAGCTTGCTCTAGAACGCTCAATAAACTCTCACAACCG-3′（SEQ?ID?NO.5）
AI4-R:5′-TCTAGACCAAGCTTTGACTATGATTGACCCTGTCTGAG-3′（SEQ?ID?NO.6）
PCR reaction: the pNDV/AI4 plasmid of take is formulated as follows the PCR reaction system as template:
| 10×High?Fidelity?Buffer | 2.5μL |
| Primer?AI4-F(50μmol/L) | 0.5μL |
| Primer?AI4-R(50μmol/L) | 0.5μL |
| dNTPs(10mmol/L) | 1μL |
| Expand?High?Fidelity?Polymerase(3.5U/μL) | 0.5μL |
| pNDV/AI4 | 2μL |
| Mg 2+ | 0.5μL |
| SW | 17.5μL |
PCR reaction cycle parameter: 94 ℃ of denaturation 5min, 94 ℃ of 50s, 60 ℃ of 1min30s, 72 ℃ of extensions, the extension time is 1min/kb, 15 rear 72 ℃ of extension 10min of circulation, 4 ℃ of preservations.
3, the structure of plasmid pUCAI4-PM-S1
Plasmid pUCAI4-PM is cut to rear recovery with Apa I enzyme, spend the phosphorylation agent box and carry out the dephosphorylation processing.After plasmid pCR2.1-S1 is cut with Apa I enzyme, reclaim the purpose fragment and be connected with the carrier after dephosphorylation, be converted into the bacillus coli DH 5 alpha competent cell, extract plasmid, positive plasmid called after pUCAI4-PM-S1.
4, the acquisition of full-length clone pNDV/AI4-S1
After plasmid pUCAI4-PM-S1 and pNDV/AI4 carry out double digestion by Age I and BstZ17 I simultaneously, reclaim the small segment after plasmid pUCAI4-PM-S1 enzyme is cut, the large fragment of cutting rear recovery with the pNDV/AI4 enzyme is connected, positive colony called after pNDV/AI4-S1.
Above each toolenzyme is purchased from MBI Fermentas.
Step 2: the rescue of recombinant virus rAI4-S1 strain
Biomaterial is prepared
But the BSR-T7/5 cell of stably express t7 rna polymerase: be so kind as to give by the step researcher of CHIGO of Harbin Veterinary Medicine Inst., China Academy of Agriculture.The CEF cell is by laboratory preparation voluntarily according to a conventional method.
Eukaryon expression plasmid pCI-NP, pCI-P, pCI-L(Hu Shunlin, Yanmei ZHANG, Sun Qing, Liu Yuliang etc. the foundation [J] of goose source Avian pneumo-encephalitis virus rescue system. microbiology circular .2007,34 (3)): build, preserve by the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory.Wherein, eukaryon expression plasmid pCI-NP, the pCI-P of expression NP and P gene also are disclosed in (the good .(2005 of Liu Yu). and produce infectious ZJ1 strain goose source newcastle disease from the cDNA clone); The eukaryon expression plasmid pCI-L that expresses the L gene also is disclosed in (Hu Shunlin .(2007). foundation and the application thereof of goose source Avian pneumo-encephalitis virus Reverse Genetics platform).
SPF kind egg, purchased from Beijing Cimmeria company, is hatched in the Ministry of Agriculture of Yangzhou University livestock and poultry loimology emphasis open laboratory.
1, the rescue of recombinant virus
Plasmid used during transfection (pNDV/AI4-S1, pCI-NP, pCI-P and pCI-L) all adopts QIAprep SpinMiniPrepKit extracting.By pCI-NP, pCI-P and pCI-L3 helper plasmid and the NDV full length cDNA clone pNDV/AI4-S1 cotransfection BSR-T7/5 cell of recombinating, adding final concentration after 18-24h is the 10%SPF embryo allantoic liquid, after 60h by after transfection sample freeze thawing 1 time, gather in the crops and inoculate 9～11 age in days SPF chicken embryos, collect chick embryo allantoic liquid, press OIE standard test hemagglutinative titer, a blind passage generation, obtain the recombinant vaccine strain rAI4-S1 that expresses infectious bronchitis virus S1 gene.
On the chicken embryo, go down to posterity after 5 times, the HA that surveys tires as 9log
2, compare with maternal strain AI4 1 titre that descended, the blood clotting characteristic can be suppressed by the monoclonal antibody specific for NDV HN albumen.
2, RT-PCR verifies the viral rAI4-S1 that is rescued
Hemagglutination test (HA test) (hemagglutination, HA) and red cell agglutination suppress experiment (hemagglutination inhibition, HI) detect all positive allantoic fluid, after SPF chicken embryo uploaded for 4 generations, collect total RNA of allantoic fluid extracting virus, react for RT-PCR after becoming cDNA with the reverse transcription of 6nt random primer.
With primer S1-F and S1-R amplification S1 gene, length is about 1700bp, reclaims the evaluation of check order of purpose fragment, and the S1 gene of the sequencing result demonstration infectious bronchitis strain (Jiangsu strain isolated CK/CH/JS/06 II) of amplified fragments successfully inserts.
Step 3: the Identification of Biological Characteristics of recombinant virus
1, the mensuration of MDT and ICPI
The average lethal time of chicken embryo (mean death time, MDT) is measured: with sterile saline, recombinant virus rAI4-S1 is done respectively to 10 times (10
-6, 10
-710
-10) gradient dilution, 5 piece of 9～11 age in days SPF chicken embryo of each extent of dilution inoculation, every embryo 0.1mL arranges the chicken embryo of 5 pieces of inoculation physiological saline in contrast simultaneously.Hatch for 37 ℃, discard chicken embryo dead in 24h, observe once until 120h calculates viral MDT by the OIE standard method every 12h afterwards.Result: a virus 5 dilution inoculated into chick embryo does not have death in 120h, and the MDT value of this strain is greater than 120h.
Chick intracranial inoculation pathogenic index (intracerebral pathogenicity index, ICPI) measure: get fresh viral allantoic fluid that the HA titre is greater than 4log2 after steriling test with not containing the 10 times of dilutions of reforming of the physiological saline of microbiotic sterilizing, SPF chicken through 10 1 ages in days of intracranial inoculation, every intracranial inoculation 0.05mL arranges the SPF chicken of 10 injecting normal salines in contrast simultaneously.By the OIE standard method, chicken is given a mark, calculated viral ICPI.Allantoic fluid is all not morbidities or dead after intracranial inoculation 1 age in days SPF chicken, and ICPI is 0, and the viral virulence that shows to be rescued meets weak malicious standard fully.
2, cell infection test
By inoculated into chick embryo inoblast after recombinant virus rAI4-S1 and maternal viral AI4 dilution, the NDV chicken serum of take respectively after infection 48h resists more or the IBV chicken serum resists as primary antibodie more, the anti-chicken IgG of the rabbit of FITC mark is the two anti-indirect immunofluorescence assay (immufluorescence assay, IFA) that carry out.
Result: in the recombinant virus-infected cell hole, all can observe the specificity fluorescent signal; Maternal virus infected cell hole can detect fluorescence with how anti-NDV serum is as primary antibodie, and other can not detect fluorescence.Recombinant virus and maternal virus infected cell be take all negative (see figure 2)s of fluoroscopic examination result that healthy SPF chicken negative serum is primary antibodie.
Claims (2)
1. express the recombinant Newcastle disease vaccine strain rAI4-S1 of S 1 protein of infectious bronchitis virus, its preserving number CGMCC No:8054.
2. the construction process of the recombinant Newcastle disease vaccine strain rAI4-S1 of the described expression S 1 protein of infectious bronchitis virus of claim 1, it is characterized in that: the genome total length transcription vector pNDV/AI4 that the gene VII type epidemic strain NDV of take causes weak malicious AI4 strain is skeleton, between the P of the insertion of the sequence shown in SEQ ID NO.7 AI4 pnca gene group total length transcription vector pNDV/AI4, M gene, through Reverse Genetics, obtain recombinant virus rAI4-S1.
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| CN109985235A (en) * | 2019-01-29 | 2019-07-09 | 苏州世诺生物技术有限公司 | Infectious bronchitis of chicken genetic engineering subunit vaccine |
| CN112626123A (en) * | 2020-12-31 | 2021-04-09 | 华农（肇庆）生物产业技术研究院有限公司 | Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof |
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| CN113462660A (en) * | 2021-07-22 | 2021-10-01 | 武汉大学 | Recombinant Newcastle disease vector vaccine for expressing avian infectious bronchitis virus S protein, preparation method and application |
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| CN104195154A (en) * | 2014-07-31 | 2014-12-10 | 哈尔滨医科大学 | Reverse genetic operation system of Newcastle disease virus Mukteswar medium-toxicity vaccine strain and application of reverse genetic operation system |
| CN104195154B (en) * | 2014-07-31 | 2016-07-06 | 哈尔滨医科大学 | Avian pneumo-encephalitis virus Mukteswar mesogenic vaccine strain reverse genetic operating system and application thereof |
| CN106390112A (en) * | 2016-08-31 | 2017-02-15 | 天津瑞普生物技术股份有限公司 | Preparation method for triple inactivated vaccine of recombinant Newcastle disease, bird flu and infectious bronchitis |
| CN108728419A (en) * | 2018-06-07 | 2018-11-02 | 扬州大学 | Express aviadenovirus penton Protein reconstitutions newcastle disease vaccine Candidate Strain rAI4-penton and construction method |
| CN109985235A (en) * | 2019-01-29 | 2019-07-09 | 苏州世诺生物技术有限公司 | Infectious bronchitis of chicken genetic engineering subunit vaccine |
| CN112626123A (en) * | 2020-12-31 | 2021-04-09 | 华农（肇庆）生物产业技术研究院有限公司 | Recombinant plasmid, recombinant gene VII type Newcastle disease virus and culture method thereof |
| CN112852758A (en) * | 2021-02-07 | 2021-05-28 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Recombinant Newcastle disease virus for expressing avian infectious bronchitis virus S protein and preparation method and application thereof |
| CN113462660A (en) * | 2021-07-22 | 2021-10-01 | 武汉大学 | Recombinant Newcastle disease vector vaccine for expressing avian infectious bronchitis virus S protein, preparation method and application |
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