CN106085970A - The heat-resisting vaccine strain of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that expression signal peptide is replaced and preparation method - Google Patents

The heat-resisting vaccine strain of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that expression signal peptide is replaced and preparation method Download PDF

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CN106085970A
CN106085970A CN201610426943.XA CN201610426943A CN106085970A CN 106085970 A CN106085970 A CN 106085970A CN 201610426943 A CN201610426943 A CN 201610426943A CN 106085970 A CN106085970 A CN 106085970A
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邵华斌
温国元
罗青平
潘兹书
乔磊
罗玲
王红琳
张腾飞
汪宏才
张蓉蓉
卢琴
艾地云
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Institute of Animal Science and Veterinary of Hubei Academy of Agricultural Sciences
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Abstract

The invention discloses the heat-resisting vaccine strain of recombinant Newcastle disease and the preparation method of the H5 subtype avian influenza HA albumen that a kind of expression signal peptide is replaced, described heat-resisting vaccine strain Classification And Nomenclature is rTS tHA/H5, being preserved in Wuhan University's China typical culture collection center on April 19th, 2016, preserving number is CCTCC NO:V201628.This vaccine strain after infection animal or cell, can high efficient expression H5 subtype avian influenza virus truncate HA albumen, the tPAs carried can strengthen secreting, expressing and the immunogenicity of truncate HA albumen.This strain can be used for preparing newcastle disease, the heat-resisting live vaccine of H5 subtype avian influenza bigeminy.

Description

The resistance to epidemic disease due to heat pathogen of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that expression signal peptide is replaced Seedling strain and preparation method
Technical field
The invention belongs to biology field, be specifically related to the H5 subtype avian influenza HA egg that a kind of expression signal peptide is replaced The heat-resisting vaccine strain of white recombinant Newcastle disease and preparation method.
Background technology
H5 subtype avian influenza virus can cause acute fatal sexually transmitted disease--the high pathogenic avian influenza of birds, its clinical table It is now high mortality, egg drop reduction, serious respiratory symptom etc..H5 subtype avian influenza also can infect the mankind, and causes morbidity. Prophylactic immunization inactivated vaccine is the Main Means of China's current prevention and control H5 subtype avian influenza.Traditional inactivated vaccine preparation is simpler Single, store convenient transportation, immune effect is lasting, has played important function in safety control of bird flu.But inactivated vaccine also has Weak point, as needed intramuscular injection, time-consuming, immunity cost is high.The use of inactivated vaccine adds to a certain extent faces Distinguish the difficulty of wild virus infection and vaccine immunity on bed, and there is scattered poison risk.Therefore, H5 cheap, safe and efficient is researched and developed sub- Type avian influenza vaccine has important practical significance.
Along with the continuous progress of Protocols in Molecular Biology, the research and development of novel gene engineered vaccine constantly make a breakthrough, wherein Novel multi-connected live vaccine based on Newcastle Disease poisonous carrier is one of research and development focus, and the immunogenic gene of many cause of diseases exists Successful expression in newcastle carrier, and achieve preferable immune protective effect, as infectious bursa of Fabricius virus VP2 albumen, The HA albumen of H5 subtype avian influenza virus, the G glycoprotein of rabies virus, the CoV albumen of SARS virus, people The Gag albumen etc. of para-immunity defective virus.This type of vaccine has inducing systemic immunity (humoral immunization, cellular immunization and viscous Film immunity), high Embryo Gallus domesticus growth characteristics, low production cost, immunization ways easy modes such as () drinking-water, aerosol, eye dripping/collunariums, The advantages such as safety (occurring restructuring and virulence to return strong probability between strain minimum).But existing Avian pneumo-encephalitis virus carrier bacterin is all With nonrefractory type Avian pneumo-encephalitis virus as framework construction.
The immunogenicity of vaccine relies primarily on antigen levels and antigen presentation efficiency.For in theory, it is positioned Cytoplasm Antigen can more effectively induce immunoreation.Tissue-typed plasminogen activator signal sequence (tPAs) be one special Albumen sorting signals, it directly sorts the antigen expressed to endoplasmic reticulum.With tPAs merge after, Japanese B encephalitis virus and Expression in the envelope protein of bovine viral diarrhea virus cell after transfection is significantly higher than wild-type protein, and main On Cytoplasm to be positioned and cell membrane, higher humoral immunization and cellular immunization can be caused.
The existing H5 avian influenza that listed, newcastle bigeminy vaccine are all by H5 subtype avian influenza virus strain and newcastle Being prepared from after Strain inactivation, it is primarily due to H5 subtype avian influenza virus and has certain pathogenic, it is impossible to For preparing live vaccine.The shortcoming of this type of vaccine is to need intramuscular injection, time-consuming, and immunity cost is high.The use of inactivated vaccine Add the difficulty distinguishing wild virus infection and vaccine immunity clinically to a certain extent, and there is scattered poison risk.
The document that number of patent application is " CN200680000024.0 " discloses expression H_5 subtype HA albumen Recombinant Newcastle disease virus LaSota attenuated vaccine strain.This vaccine strain does not possess significant heat-resistant quality, and uses HA egg The signal peptide of Bai Zishen.
Therefore, how to provide a kind of and there is the H5 that expression signal peptide heat-resisting, that stability strong, refrigerated condition requirement is low is replaced The heat-resisting vaccine strain of recombinant Newcastle disease of subtype avian influenza HA albumen and preparation method are the skills that those skilled in the art are urgently to be resolved hurrily Art problem.
Summary of the invention
For deficiency of the prior art, the present invention conducts in-depth research, it is provided that a kind of expression signal peptide is replaced The heat-resisting vaccine strain of recombinant Newcastle disease of H5 subtype avian influenza HA albumen and preparation method.
The recombinant Newcastle disease that one aspect of the present invention relates to the H5 subtype avian influenza HA albumen that a kind of expression signal peptide is replaced is resistance to Hot vaccine strain, it is characterised in that described heat-resisting vaccine strain Classification And Nomenclature is rTS-tHA/H5, was preserved on April 19th, 2016 Wuhan University's China typical culture collection center, preserving number is CCTCC NO:V201628.
The recombinant Newcastle disease that another aspect of the present invention further relates to the H5 subtype avian influenza HA albumen that expression signal peptide is replaced is resistance to The preparation method of hot vaccine strain, it is characterised in that comprise the steps:
The PCR amplification of 1.1 H5 HA Gene of H 9 Subtype AIVs
H5N1 bird flu virus A/chicken/Hubei/489/2004 strain is bred on 9-11 day instar chicken embryo, connects Chick embryo allantoic liquid is gathered in the crops after planting 5 days；The allantoic fluid of results carries out the RT-PCR amplification of HA gene, and amplimer is: upstream is drawn Thing 5 '-AGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGC GAAATCTCTTCAATTT GCATTGGTTACCATGC-3 ', downstream primer 5 '-ATCGGGGCACTCCGATTCTACCCGTATTTTTTCTTAATTATTATTA TCCTCTCTTTTTTC-3′；PCR primer carrying out agarose gel reclaim after purification, measure DNA concentration, pending clone is even Connect；
The PCR amplification of 1.2 Avian pneumo-encephalitis virus heat-resistant carriers fragments
Obtaining Avian pneumo-encephalitis virus carrier sequence with the method for super LA-PCR, amplimer is: forward primer 5 '- TCGGAGTGCCCCGATTGTGCCAAGATGGACTCATC-3 ', downstream primer 5 '-AGCAGAGCCCTCTCTTCATTGCATCCA TGGTGGCAAGCTTTCTACCCGTATTTTTTCTTAATCCTTAATA-3′；Amplification template is Avian pneumo-encephalitis virus heat-resisting strain plasmid PTS09-C, carries out PCR primer reclaiming purification, measures its concentration, treats that clone connects；
The clone of 1.3 recombiant plasmid pTS-tHA/H5 connects
Use In-fusion to clone interconnection technique, HA1 genetic fragment good for purification and heat-resistant carriers fragment are cloned Connecting, connect product and convert DH5 α competent cell, the cell after conversion is chosen single bacterium colony and is entered after cultivating 16 hours on LB plate Row liquid culture, the PCR that bacterium solution carries out HA1 gene identifies, the bacterium solution being accredited as the positive carries out plasmid extraction；
The rescue of 1.4 recombinant virus rTS-tHA/H5 recovers
Use lipofection, the recombiant plasmid pTS-tHA/H5 built and three are expressed NP, P and L egg respectively White helper plasmid cotransfection BHK-21 cell, transfects latter 72 hours, multigelation harvesting liquid, inoculates 9-11 age in days SPF Embryo Gallus domesticus.Inoculate latter 5 days, gather in the crops chick embryo allantoic liquid, the weight of the H5 subtype avian influenza HA albumen that isolated expression signal peptide is replaced The group heat-resisting vaccine strain of newcastle.
The invention still further relates to the resistance to epidemic disease due to heat pathogen of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that above-mentioned expression signal peptide is replaced Seedling strain application in preparing recombinant virus.
The invention still further relates to the resistance to epidemic disease due to heat pathogen of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that above-mentioned expression signal peptide is replaced Seedling strain application in preparing H5 avian influenza, newcastle bigeminy heat-resisting live vaccine vaccine.
This application discloses the resistance to epidemic disease due to heat pathogen of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that a kind of expression signal peptide is replaced Miao Zhu, this vaccine strain has significant heat-resistant quality, can greatly reduce the dependence to cold chain system, save preserve cost of transportation and Improve vaccine heat stability.This strain, can the HA1 egg of high efficient expression H5 subtype avian influenza virus after infection animal or cell In vain, the tPAs carried can strengthen secreting, expressing and the immunogenicity of HA1 albumen.This strain can be used for preparing H5 avian influenza, new city The heat-resisting live vaccine of epidemic disease bigeminy.
Accompanying drawing explanation
Fig. 1 is the recombinant heat-proof newcastle of the H5 subtype avian influenza virus truncate HA albumen that expression signal peptide of the present invention is replaced The structure schematic diagram of virus full length plasmid.Wherein rTS09-C is the genome structure of parent plant, and rTS-tHA/H5 is at rTS09- C genome inserts the structure after truncate HA albumen, is labelled with the overall sequence composition of insertion sequence in detail.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described further, the following stated, is only the preferable enforcement to the present invention Example, not does the restriction of other forms to the present invention, and any those skilled in the art are possibly also with the disclosure above Technology contents be changed to the Equivalent embodiments that changes on an equal basis.Every without departing from the present invention program content, according to the present invention Technical spirit any simple modification that following example are done or equivalent variations, the most within the scope of the present invention.
Embodiment 1
The first step, the structure of recombinant full-lenght plasmid pTS-tHA/H5 and qualification
The PCR amplification of 1.1 H5 HA Gene of H 9 Subtype AIVs
H5N1 bird flu virus A/chicken/Hubei/489/2004 strain is bred on 9-11 day instar chicken embryo, connects Chick embryo allantoic liquid is gathered in the crops after planting 5 days.The allantoic fluid of results carries out the RT-PCR amplification of HA gene, and amplimer is: upstream is drawn Thing 5 '-AGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGC GAAATCTCTTCAATTT GCATTGGTTACCATGC-3 ', downstream primer 5 '-ATCGGGGCACTCCGATTCTACCCGTATTTTTTCTTAATTATTATTA TCCTCTCTTTTTTC-3 ' (underscore part is the complementary series connected for In-fusion clone).Agarose gel electrophoresis Testing goal band about 1.1kb, be consistent with intended 1101bp (the HA1 mrna length deleting native signal peptide is 987bp, point Do not introduce gene start sequence and gene termination sequence in HA1 gene front-end and back-end, introduce in HA1 gene front end simultaneously Kozak sequence and tPAs sequence).PCR primer carries out agarose gel reclaim after purification, measure DNA concentration, pending clone Connect.
The PCR amplification of 1.2 Avian pneumo-encephalitis virus heat-resistant carriers fragments
Obtaining Avian pneumo-encephalitis virus carrier sequence with the method for super LA-PCR, amplimer is: forward primer 5 '- TCGGAGTGCCCCGATTGTGCCAAGATGGACTCATC-3 ', downstream primer 5 '-AGCAGAGCCCTCTCTTCATTGCATCCA (underscore part is to clone for In-fusion to TGGTGGCAAGCTTTCTACCCGTATTTTTTCTTAATCCTTAATA-3 ' The complementary series connected).Amplification template is Avian pneumo-encephalitis virus heat-resisting strain plasmid pTS09-C.Agarose gel electrophoresis testing goal Band about 18kb, is consistent with intended 17.8kb size.Carry out PCR primer reclaiming purification, measure its concentration, treat that clone is even Connect.
The clone of 1.3 recombiant plasmid pTS-tHA/H5 connects
According to the mode shown in Fig. 1, In-fusion is used to clone interconnection technique, by HA1 genetic fragment good for purification and resistance to Heat carrier fragment carries out clone and connects (catenation sequence is AGCGAAATCTCTTCA and TCGGAGTGCCCCGAT).Connect product to turn Changing DH5 α competent cell, the cell after conversion is chosen single bacterium colony and is carried out liquid culture after cultivating 16 hours on LB plate.To bacterium Liquid carries out the PCR of HA1 gene to be identified.The bacterium solution being accredited as the positive carries out plasmid extraction, treats that enzyme action and order-checking are identified.
The enzyme action of 1.4 recombiant plasmid pTS-tHA/H5 is identified with order-checking
With BamHI and MluI restriction endonuclease, total length recombiant plasmid is carried out enzyme action qualification respectively, all obtained being consistent with expection Restriction enzyme mapping.Enzyme action is identified, and correct recombiant plasmid pTS-tHA/H5 delivers to order-checking company and carries out sequencing, result table The HA1 gene of bright H5 subtype avian influenza is inserted between P and the M gene of Avian pneumo-encephalitis virus heat-resistant carriers, the success of tPAs sequence Substituted for the signal peptide sequence of HA albumen self, and all sequences is completely the same with expection, recombiant plasmid pTS-tHA/H5 builds Success.
Second step, recombinant virus rTS-tHA/H5 rescue recovers and identifies
The rescue of 2.1 recombinant virus rTS-tHA/H5 recovers
Use lipofection, the recombiant plasmid pTS-tHA/H5 built and three are expressed NP, P and L egg respectively White helper plasmid cotransfection BHK-21 cell (infecting the vaccinia virus expressing t7 rna polymerase in advance).Transfect latter 72 hours, Multigelation harvesting liquid, inoculates 9-11 age in days SPF Embryo Gallus domesticus.Inoculate latter 5 days, gather in the crops chick embryo allantoic liquid, pending newcastle Viral diagnosis.
The hemagglutinative titer of 2.2 recombinant viruses and fluorescence quantitative PCR detection
With hemagglutination test and the method for quantitative fluorescent PCR, detecting results chick embryo allantoic liquid, result is newcastle Positive.Tentatively show that recombinant virus rTS-tHA/H5 saves successfully.
PCR amplification and the order-checking of 2.3 recombinant virus HA1 genes
The specificity amplification primer of application H5 subtype avian influenza virus HA1 gene, the HA1 to recombinant virus rTS-tHA/H5 Gene carries out RT-PCR amplification, and pcr amplification product is carried out Sequence analysis, obtains the HA1 gene order of 1101bp length (SEQ ID No.1), holds from 5 ' to 3 ', contains the gene end of the part non-coding area sequence (UTR) of P gene, P gene successively Only sequence (GE), intermediate sequence (IG), the gene start sequence (GS) of HA1 gene, Kozak sequence, tPAs sequence, HA1 gene Sequence (deleting native signal peptide), the dual termination codon of HA1 gene, the GE sequence of HA1 gene, IG sequence, M gene GS sequence and the UTR sequence of M gene.Consistent with expected sequence 100%.
The H5 subtype avian influenza virus truncate HA albumen that 3rd step, signal peptide are replaced is in recombinant virus rTS-tHA/H5 Express checking
The expression of the HA1 albumen that 3.1 indirect immunofluorescence detection signal peptides are replaced
Recombinant virus rTS-tHA/H5 and rTS09-C (comparison) is infected BHK-21 cell with 0.01MOI respectively, after 24h Carrying out indirect immunofluorescene assay, one resists for NDV positive chicken serum or H5 subtype avian influenza positive serum.Testing result shows, It is intracellular that recombinant virus rTS-tHA/H5 infects, and uses NDV positive chicken serum or H5 subtype avian influenza positive serum as one Green fluorescence all can be detected time anti-, and the cell that rTS09-C strain is infected only detects fluorescence under NDV positive serum effect.
The expression of the HA albumen that 3.2 Western Blot method detection signal peptides are replaced
Recombinant virus rTS-tHA/H5 and rTS09-C (comparison) is infected BHK-21 cell with 0.01MOI respectively, after 24h Collect cell pyrolysis liquid, be one anti-to carry out Western Blot detection with H5 subtype avian influenza positive chicken serum.Testing result table Bright, an obvious purpose band occurs near 40KDa, close with intended 40.6KDa size.Show what signal peptide was replaced HA1 albumen is in the intracellular correct expression of recombinant virus infection.
4th step, the Identification of Biological Characteristics of recombinant virus rTS-tHA/H5
The multiplication characteristic of 4.1 recombinant viruses measures
For the growing multiplication situation of heavier papova rTS-tHA/H5 strain Yu parent's rTS09-C strain, two strain virus are divided Not with 100 TCID50Inoculation BHK-21 cell, after inoculation, 24h, 48h, 72h, 96h take supernatant 500 μ l, measure virus TCID50, Draw the growth curve of virus.Result shows, recombinant virus rTS-tHA/H5 strain and parent's rTS09-C strain are at BHK-21 cell Growth curve is similar, and viral titer is close, and about 107.0TCID50/ml。
The Pathogenicity of 4.2 recombinant viruses
Measure the recombinant virus median lethal time (MDT/MLD) to the minimal lethal dose of Embryo Gallus domesticus, result display difference Dilution factor (10-1To 10-9) virus inoculation Embryo Gallus domesticus after 120h in all will not be lethal to Embryo Gallus domesticus, recombinant virus rTS-tHA/H5 MDT/MLD more than 120h, show that recombinant virus maintains the low toxicity characteristic of parent plant, the insertion of exogenous gene does not change weight Papova pathogenic.
The heat-resistant quality of 4.3 recombinant viruses measures
Measure recombinant virus HA heat-resistant quality under 56 DEG C of environment, nonrefractory strain LaSota strain and heat-resisting strain are set simultaneously RTS09-C strain is comparison.As shown in table 1, nonrefractory strain LaSota is after 56 DEG C of 9min of resistance to heat treatment, and HA titer reduces to 0, parent Strain rTS09-C and recombinant virus rTS-tHA/H5 still has hemagglutination activity after 56 DEG C of 150min of resistance to heat treatment.Show restructuring disease The heat-resistant quality of poison rTS-tHA/H5 is consistent with parent plant, is heat-resisting strain.(the preservation of this Strain, is deposited in Wuhan big Learning China typical culture collection center, preserving number is CCTCC NO:V201628).
The HA heat-resistant quality measurement result of table 1. recombinant virus
aRepresent that virus has hemagglutination activity,bRepresent that virus does not has hemagglutination activity
Embodiment described above is the preferred embodiments of the present invention, and does not limit the practical range of the present invention.Therefore, all It is according to the equivalent modifications done by the essence of present invention, all should belong within the scope of the present invention.
<110>Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences
<120>expression signal peptide is replaced the heat-resisting vaccine strain of recombinant Newcastle disease of H5 subtype avian influenza HA albumen and preparation method
<160>1
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ttggaacatc aacactaaac cagagattgg taccaaaaat agctactaga tccaaagtaa 720
acgggcaaag tggaaggatg gagttcttct ggacaatttt aaaaccgaat gatgcaatca 780
atttcgagag taatggaaat ttcattgctc cagaatatgc atacaaaatt gtcaagaaag 840
gggactcagc aattatgaaa agtgaattgg aatatggtaa ctgcaacacc aagtgtcaaa 900
ctccaatggg ggcgataaac tctagtatgc cattccacaa catacaccct ctcaccatcg 960
gggaatgccc caaatatgtg aaatcaaaca gattagtcct tgcgactggg ctcagaaata 1020
gccctcaaag agagagaaga agaaaaaaga gaggataata ataattaaga aaaaatacgg 1080
gtagaatcgg agtgccccga t 1101
<212> Type : DNA
<211> Length : 1101
SequenceName : 1
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
agagagggct ctgctgtgtg ctgctgctgt gtggagcagt cttcgtttcg cccagcgaaa 60
tctcttcaat ttgcattggt taccatgc 88
<212> Type : DNA
4
<211> Length : 88
SequenceName : 2
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
atcggggcac tccgattcta cccgtatttt ttcttaatta ttattatcct ctcttttttc 60
<212> Type : DNA
<211> Length : 60
SequenceName : 3
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
tcggagtgcc ccgattgtgc caagatggac tcatc 35
5
<212> Type : DNA
<211> Length : 35
SequenceName : 4
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
agcagagccc tctcttcatt gcatccatgg tggcaagctt tctacccgta ttttttctta 60
atccttaata 70
<212> Type : DNA
<211> Length : 70
SequenceName : 5
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
6
agcgaaatct cttca 15
<212> Type : DNA
<211> Length : 15
SequenceName : 6
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
tcggagtgccccgat
<212> Type : DNA
<211> Length : 15
SequenceName : 7
SequenceDescription :

Claims (4)

1. the heat-resisting vaccine strain of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that expression signal peptide is replaced, it is characterised in that described Heat-resisting vaccine strain Classification And Nomenclature be rTS-tHA/H5, be preserved in Wuhan University's Chinese Typical Representative culture on April 19th, 2016 Preservation center, preserving number is CCTCC NO:V201628.
2. the preparation method of the heat-resisting vaccine strain of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that expression signal peptide is replaced, it is special Levy and be to comprise the steps:
The PCR amplification of 1.1 H5 HA Gene of H 9 Subtype AIVs
H5N1 bird flu virus A/chicken/Hubei/489/2004 strain is bred on 9-11 day instar chicken embryo, inoculates 5 Chick embryo allantoic liquid is gathered in the crops after it；The allantoic fluid of results carries out the RT-PCR amplification of HA gene, and amplimer is: forward primer 5′-AGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCGAAATCTCTTCAATTTGC ATTGGTTACCATGC-3 ', downstream primer 5 '-ATCGGGGCACTCCGATTCTACCCGTATTTTTTCTTAATTATTATTATC CTCTCTTTTTTC-3′；PCR primer carrying out agarose gel reclaim after purification, measure DNA concentration, pending clone connects；
The PCR amplification of 1.2 Avian pneumo-encephalitis virus heat-resistant carriers fragments
Obtaining Avian pneumo-encephalitis virus carrier sequence with the method for super LA-PCR, amplimer is: forward primer 5 '- TCGGAGTGCCCCGATTGTGCCAAGATGGACTCATC-3 ', downstream primer 5 '-AGCAGAGCCCTCTCTTCATTGCATCCA TGGTGGCAAGCTTTCTACCCGTATTTTTTCTTAATCCTTAATA-3′；Amplification template is Avian pneumo-encephalitis virus heat-resisting strain plasmid PTS09-C, carries out PCR primer reclaiming purification, measures its concentration, treats that clone connects；
The clone of 1.3 recombiant plasmid pTS-tHA/H5 connects
Use In-fusion to clone interconnection technique, HA1 genetic fragment good for purification and heat-resistant carriers fragment are carried out clone even Connecing, catenation sequence is AGCGAAATCTCTTCA and TCGGAGTGCCCCGAT, connects product and converts DH5 α competent cell, converts After cell cultivate 16 hours on LB plate after choose single bacterium colony and carry out liquid culture, bacterium solution is carried out the PCR mirror of HA1 gene Fixed, the bacterium solution being accredited as the positive carries out plasmid extraction；
The rescue of 1.4 recombinant virus rTS-tHA/H5 recovers
Use lipofection, the recombiant plasmid pTS-tHA/H5 built and three are expressed NP, P and L albumen respectively Helper plasmid cotransfection BHK-21 cell, transfects latter 72 hours, multigelation harvesting liquid, inoculates 9-11 age in days SPF Embryo Gallus domesticus. Inoculate latter 5 days, gather in the crops chick embryo allantoic liquid, the restructuring new city of the H5 subtype avian influenza HA albumen that isolated expression signal peptide is replaced The heat-resisting vaccine strain of epidemic disease.
3. the heat-resisting vaccine strain of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that the expression signal peptide described in claim 1 is replaced Application in preparing recombinant virus.
4. the heat-resisting vaccine strain of recombinant Newcastle disease of the H5 subtype avian influenza HA albumen that the expression signal peptide described in claim 1 is replaced Application in preparing H5 avian influenza, the heat-resisting live vaccine of newcastle bigeminy.
CN201610426943.XA 2016-06-15 2016-06-15 The heat-resisting vaccine strain of recombinant Newcastle disease and preparation method of the H5 subtype avian influenza HA albumen of expression signal peptide replacement Expired - Fee Related CN106085970B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109096377A (en) * 2018-09-19 2018-12-28 天康生物股份有限公司 Avian flu virus hemagglutinin antigen and BHK-21 cell strain, preparation method and the vaccine for expressing it
CN112126629A (en) * 2020-08-19 2020-12-25 湖北省农业科学院畜牧兽医研究所 Heat-resistant Newcastle disease virus mutant strain 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

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1942578A (en) * 2005-09-02 2007-04-04 中国农业科学院哈尔滨兽医研究所 Low virulent strain of recombinant newcastle disease lasota vaccine expressing HA protein of avian influenza-H5 virus
CN101892248A (en) * 2010-04-29 2010-11-24 王世霞 Monoclonal antibodies of avian influenza H5HA antigens
CN104059942A (en) * 2013-03-20 2014-09-24 湖北省农业科学院畜牧兽医研究所 Newcastle disease virus heat resistant live vaccine vector system and application thereof
CN105420261A (en) * 2015-10-09 2016-03-23 湖北省农业科学院畜牧兽医研究所 Newcastle disease virus heat-resistant transformation method and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1942578A (en) * 2005-09-02 2007-04-04 中国农业科学院哈尔滨兽医研究所 Low virulent strain of recombinant newcastle disease lasota vaccine expressing HA protein of avian influenza-H5 virus
CN101892248A (en) * 2010-04-29 2010-11-24 王世霞 Monoclonal antibodies of avian influenza H5HA antigens
CN104059942A (en) * 2013-03-20 2014-09-24 湖北省农业科学院畜牧兽医研究所 Newcastle disease virus heat resistant live vaccine vector system and application thereof
CN105420261A (en) * 2015-10-09 2016-03-23 湖北省农业科学院畜牧兽医研究所 Newcastle disease virus heat-resistant transformation method and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GUOYUAN WEN等: "Molecular basis for the thermostability of Newcastle disease virus", 《SCIENTIFIC REPORTS》 *
张春华等: "禽流感HA 基因密码子优化的DNA疫苗构建及其免疫原性的初步研究", 《中国医药生物技术》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109096377A (en) * 2018-09-19 2018-12-28 天康生物股份有限公司 Avian flu virus hemagglutinin antigen and BHK-21 cell strain, preparation method and the vaccine for expressing it
CN109096377B (en) * 2018-09-19 2021-03-23 天康生物股份有限公司 Avian influenza virus hemagglutinin antigen, BHK-21 cell strain expressing avian influenza virus hemagglutinin antigen, preparation method and vaccine
CN112126629A (en) * 2020-08-19 2020-12-25 湖北省农业科学院畜牧兽医研究所 Heat-resistant Newcastle disease virus mutant strain 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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