CN105969741A - Recombinant Newcastle disease heat-resisting vaccine strain co-expressing HA and chicken interleukin 6 proteins of H9 subtype avian influenza virus and preparation method thereof - Google Patents
Recombinant Newcastle disease heat-resisting vaccine strain co-expressing HA and chicken interleukin 6 proteins of H9 subtype avian influenza virus and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a recombinant Newcastle disease heat-resisting vaccine strain co-expressing HA and chicken interleukin 6 proteins of an H9 subtype avian influenza virus and a preparation method thereof. The heat-resisting vaccine strain is classified and named as rTS-HA/H9-IL6, is preserved, with CCTCC NO: V201630, at the China Center for Type Culture Collection of Wuhan University on April 19, 2016. After animals or cells are infected with the vaccine strain, the full-length HA protein and the chicken interleukin 6 protein of the H9 subtype avian influenza virus can be efficiently expressed. The strain can be used for preparation of H9 avian influenza and a Newcastle disease bivalent heat-resisting live vaccine, and the chicken interleukin 6 protein plays the effects of an immune enhancer.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of coexpression H9 subtype avian influenza virus HA and chicken is situated between in vain
The heat-resisting vaccine strain of recombinant Newcastle disease of element 6 albumen and preparation method.
Background technology
H9 subtype avian influenza is an important avian influenza virus subtype of harm China aviculture, belongs to low pathogenicity fowl stream
Sense.Owing to its pathogenicity is relatively low, the mortality of fowl group is the most directly caused easily to be ignored by people.But this subtype avian influenza is sick
Poison can destroy the immune system of chicken group, may result in immunosuppressant, then cause the infection of other cause of diseases such as escherichia coli time serious,
Thus causing higher mortality rate, the laying hen mortality rate infecting H9 subtype avian influenza virus is about 10%, and laying rate declines
About 20%, cause bigger economic loss.Prophylactic immunization inactivated vaccine is the main of China's current prevention and control H9 subtype avian influenza
Means.Traditional inactivated vaccine is prepared fairly simple, stores convenient transportation, and immune effect is lasting, plays in safety control of bird flu
Important function.But in place of inactivated vaccine also has some shortcomings, as needed intramuscular injection, time-consuming, immunity cost is high.Inactivation epidemic disease
The use of Seedling adds the difficulty distinguishing wild virus infection and vaccine immunity clinically to a certain extent, and there is scattered poison risk.
Therefore, research and develop H9 subtype avian influenza vaccine cheap, safe and efficient to have 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.
Research shows, some avian cytokines have adjuvant character.Interleukin-6 (IL-6) is that one is multi-functional carefully
Intracellular cytokine, it plays an important role at aspects such as regulation immune response, acute phase response and hemopoietic.Coexpression IL-6 and
The expression plasmid pGPIL-6 of the gp120 albumen of HIV 1B hypotype representative strains, finds after immune mouse that this plasmid can stimulate body
Produce cytotoxic T lymphocyte (CTL) reaction, and be remarkably improved body CD4+ and the quantity of CD8+T lymphocyte, show
Cytokine IL-6 has well played the effect of immunological adjuvant.Chicken recombinant interleukin-6 albumen (rChIL-6) is infected with chicken
Property bronchitis virus (IBDV) inactivated vaccine immunized chicks after, find rChIL-6 can raise cytokine chicken interleukin-2 4
(ChIL-4) the H9 bird flu, the newcastle bigeminy vaccine that are presented with having listed are all by H9 subtype avian influenza virus strain and new city
Being prepared from after the inactivation of epidemic disease strain, it is primarily due to H9 subtype avian influenza virus and has certain pathogenic, no
Can be used for preparing live vaccine.The shortcoming of this type of vaccine is to need intramuscular injection, time-consuming, and immunity cost is high.Making of inactivated vaccine
It is used in the difficulty that adds somewhat to distinguish wild virus infection clinically with vaccine immunity, and there is scattered poison risk.
The document that number of patent application is " CN200810222776.2 " discloses expression bird flu virus H9 subtype HA protein
Recombinant Newcastle disease virus LaSota attenuated vaccine strain.This vaccine strain does not possess significant heat-resistant quality, and does not adds immunity increasing
The strong factor.
Therefore, how to provide a kind of and there is coexpression H9 subtype avian influenza heat-resisting, that stability strong, refrigerated condition requirement is low
Virus HA and the heat-resisting vaccine strain of recombinant Newcastle disease of chicken interleukin-2 6 albumen and preparation method are that those skilled in the art are urgently to be resolved hurrily
Technical 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 coexpression H9 hypotype fowl stream
The heat-resisting vaccine strain of recombinant Newcastle disease of Influenza Virus HA and chicken interleukin-2 6 albumen and preparation method.
One aspect of the present invention relates to a kind of coexpression H9 subtype avian influenza virus HA and the restructuring new city of chicken interleukin-2 6 albumen
The heat-resisting vaccine strain of epidemic disease, it is characterised in that described heat-resisting vaccine strain Classification And Nomenclature was rTS-HA/H9-IL6, April 19 in 2016
Day is preserved in Wuhan University's China typical culture collection center, and preserving number is CCTCC NO:V201630.
Another aspect of the present invention further relates to coexpression H9 subtype avian influenza virus HA and the restructuring new city of chicken interleukin-2 6 albumen
The preparation method of the heat-resisting vaccine strain of epidemic disease, it is characterised in that comprise the steps:
The insertion of 1.1 HA/H9 genes
H9N2 bird flu virus A/Chicken/Hubei/C1/2007 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 '-CAGCTATATTAAGGATTAAGAAAAAATACGGGTAGAAAGCTTGCCACCATGGAAACAATATCACTAATAGC-
3 ', downstream primer 5 '-ATCGGGGCACTCCGATTCTACCCGTATTTTTTCTTAATTATTATTATATACAAATGTTGCATC
TGC-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 '-TCGGAGTGCCCCGATTGTGCCAAGATGGACTC-3 ', downstream primer 5 '-TCCTTAATATAGCTGAATTGATTGCAGCTG
CGCGATC-3′；Amplification template is Avian pneumo-encephalitis virus heat-resisting strain plasmid pTS09-C；Carry out PCR primer reclaiming purification, measure it
Concentration, treats that clone connects；
The connection of 1.3 recombiant plasmid pTS-HA/H9 and qualification
Use In-fusion to clone interconnection technique, HA genetic fragment good for purification and heat-resistant carriers fragment are cloned
Connect (catenation sequence is CAGCTATATTAAGGA and TCGGAGTGCCCCGAT).Connect product and convert DH5 α competent cell,
Cell after conversion is chosen single bacterium colony and is carried out liquid culture after cultivating 16 hours on LB plate；Bacterium solution is carried out the PCR of HA gene
Identifying, the bacterium solution being accredited as the positive carries out plasmid extraction and carries out enzyme action qualification；
The PCR amplification of 14 IL6 genes
With plasmid pIL6 as template, carrying out the PCR amplification of IL6 gene, amplimer is: forward primer 5 '-ACAGGGCCAAAGAAATTAGAAAAAAGTACGGGTAGAAAGCAGCTTGCCACCATGAACTTCACCGAGGGCTGCGA-
3 ', downstream primer 5 '-CTCTGAATGTCTCCCTTCTACCCGTATTTTTTCTTAATTATTATTAGGCACTGAAACTCCTGG
TC-3；PCR primer carrying out agarose gel reclaim after purification, measure DNA concentration, pending clone connects；
The PCR amplification of 1.5 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 '-GGGAGACATTCAGAGATCAGGGCGAGTCACCCGGGT-3 ', downstream primer 5 '-TTTCTTTGGCCCTGTATTGATTATTA
GTGGGTCGC-3 ', amplification template is middle interstitial granules pTS-HA/H9, carries out PCR primer reclaiming purification, measures its concentration, treat
Clone connects；
The connection of 1.6 recombiant plasmid pTS-HA/H9-IL6 and qualification
Use In-fusion to clone interconnection technique, IL6 genetic fragment good for purification and heat-resistant carriers fragment are cloned
Connect (catenation sequence is ACAGGGCCAAAGAAA and GGGAGACATTCAGAG)；Connect product and convert DH5 α competent cell,
Cell after conversion is chosen single bacterium colony and is carried out liquid culture after cultivating 16 hours on LB plate；Bacterium solution is carried out the PCR of IL6 gene
Identify；The bacterium solution being accredited as the positive carries out plasmid extraction and carries out enzyme action qualification；
The rescue of 1.7 recombinant virus rTS-HA/H9-IL6 recovers
Use lipofection, the recombiant plasmid pTS-HA/H9-IL6 built and three are expressed NP, P and L respectively
The helper plasmid cotransfection BHK-21 cell of albumen；Transfect latter 72 hours, multigelation harvesting liquid, inoculate 9-11 age in days
SPF Embryo Gallus domesticus；Inoculate latter 5 days, gather in the crops chick embryo allantoic liquid, isolated coexpression H9 subtype avian influenza virus HA and chicken interleukin-2 6
The heat-resisting vaccine strain of recombinant Newcastle disease of albumen.
The recombinant Newcastle disease that the invention still further relates to above-mentioned coexpression H9 subtype avian influenza virus HA and chicken interleukin-2 6 albumen is resistance to
The application in preparing recombinant virus of the hot vaccine strain.
The recombinant Newcastle disease that the invention still further relates to above-mentioned coexpression H9 subtype avian influenza virus HA and chicken interleukin-2 6 albumen is resistance to
The application in preparing H5 avian influenza, the heat-resisting live vaccine of newcastle bigeminy of the hot vaccine strain.
This application discloses a kind of recombinant Newcastle disease virus expressing H9 subtype avian influenza virus truncate HA albumen heat-resisting
Strain.This vaccine strain has significant heat-resistant quality, can greatly reduce the dependence to cold chain system, saves and preserves cost of transportation and carry
High vaccine heat stability.This strain, can the total length HA egg of high efficient expression H9 subtype avian influenza virus after infection animal or cell
White and chicken interleukin-2 6 albumen.This strain can be used for preparing H9 bird flu, the heat-resisting live vaccine of newcastle bigeminy, chicken interleukin-2 6 albumen
Serve the effect of immunostimulant.
Accompanying drawing explanation
Fig. 1 is that the present invention can coexpression H9 subtype avian influenza virus HA albumen and the recombinant heat-proof newcastle of chicken interleukin-2 6
The structure schematic diagram of virus full length plasmid.Wherein rTS09-C is the genome structure of parent plant, rTS-HA/H9-IL6 be
RTS09-C genome inserts the structure after HA and IL6 gene, 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-HA/H9-IL6 and qualification
Recombinant full-lenght plasmid pTS-HA/H9-IL6 is by by white to H9 HA Gene of H 9 Subtype AIV (HA/H9) and chicken
Interleukin 6 gene (IL6) is jointly inserted into and obtains in Avian pneumo-encephalitis virus recombiant plasmid pTS09-C.As it is shown in figure 1, it builds
Being divided into two to walk greatly, the first step is between P and the M gene that HA/H9 gene is inserted into plasmid pTS09-C, it is thus achieved that middle interstitial granules
pTS-HA/H9；Second step is to be inserted into before the NP gene of pTS-HA/H9 by IL6 gene, final acquisition recombinant full-lenght plasmid
pTS-HA/H9-IL6。
The insertion of 1.1 HA/H9 genes
1.1.1 the PCR amplification of H9 HA Gene of H 9 Subtype AIV
H9N2 bird flu virus A/Chicken/Hubei/C1/2007 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 '-CAGCTATATTAAGGATTAAGAAAAAATACGGGTAGAAAGCTTGCCACCATGGAAACAATATCACTAATAGC-
3 ', downstream primer 5 '-ATCGGGGCACTCCGATTCTACCCGTATTTTTTCTTAATTATTATTATATACAAATGTTGCATC
TGC-3 ' (underscore part is the complementary series connected for In-fusion clone).Agarose gel electrophoresis testing goal bar
Band about 1.8kb, being consistent with intended 1774bp, (HA1 mrna length is 1680bp, introduces in HA gene front-end and back-end respectively
Gene start sequence and gene termination sequence, introduce Kozak sequence in HA gene front end simultaneously).PCR primer is carried out agar
After sugar gel purification, measuring DNA concentration, pending clone connects.
1.1.2 the PCR amplification of Avian pneumo-encephalitis virus heat-resistant carriers fragment
Obtaining Avian pneumo-encephalitis virus carrier sequence with the method for super LA-PCR, amplimer is: forward primer 5 '-TCGGAGTGCCCCGATTGTGCCAAGATGGACTC-3 ', downstream primer 5 '-TCCTTAATATAGCTGAATTGATTGCAGCTG
CGCGATC-3 ' (underscore part is the complementary series connected for In-fusion clone).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.Right
PCR primer carries out reclaiming purification, measures its concentration, treats that clone connects.
1.1.3 the connection of recombiant plasmid pTS-HA/H9 and qualification
Use In-fusion to clone interconnection technique, HA genetic fragment good for purification and heat-resistant carriers fragment are cloned
Connect (catenation sequence is CAGCTATATTAAGGA and TCGGAGTGCCCCGAT).Connect product and convert DH5 α competent cell,
Cell after conversion is chosen single bacterium colony and is carried out liquid culture after cultivating 16 hours on LB plate.Bacterium solution is carried out the PCR of HA gene
Identify.The bacterium solution being accredited as the positive carries out plasmid extraction and carries out enzyme action qualification.Respectively with BamHI and MluI restriction endonuclease to total length weight
Group plasmid carries out enzyme action qualification, has all obtained restriction enzyme mapping in line.Enzyme action is identified correct recombiant plasmid pTS-
HA/H9 delivers to order-checking company and carries out sequencing, and result shows that the HA gene of H9 subtype avian influenza is inserted into Avian pneumo-encephalitis virus
Between P and the M gene of heat-resistant carriers, and all sequences is completely the same with expection, and middle interstitial granules pTS-HA/H9 successfully constructs.
The insertion of 1.2 IL6 genes
1.2.1 the PCR amplification of IL6 gene
Plasmid pIL6 containing chicken interleukin-2 6 gene is by this laboratory design sequence and meets at Shanghai biological engineering share public affairs
Department's synthesis.With plasmid pIL6 as template, carrying out the PCR amplification of IL6 gene, amplimer is: forward primer 5 '-ACAGGGCCAAAGAAATTAGAAAAAAGTACGGGTAGAAAGCAGCTTGCCACCATGAACTTCACCGAGGGCTGCGA-
3 ', downstream primer 5 '-CTCTGAATGTCTCCCTTCTACCCGTATTTTTTCTTAATTATTATTAGGCACTGAAACTCCTGG
TC-3 (underscore part is the complementary series connected for In-fusion clone).Agarose gel electrophoresis testing goal band
About 0.8kb, being consistent with intended 819bp, (IL6 mrna length is 726bp, introduces base in IL6 gene front-end and back-end respectively
Because of homing sequence and gene termination sequence, introduce Kozak sequence in IL6 gene front end) simultaneously.PCR primer is carried out agar
After sugar gel purification, measuring DNA concentration, pending clone connects.
1.2.2 the PCR amplification of Avian pneumo-encephalitis virus heat-resistant carriers fragment
Obtaining Avian pneumo-encephalitis virus carrier sequence with the method for super LA-PCR, amplimer is: forward primer 5 '-GGGAGACATTCAGAGATCAGGGCGAGTCACCCGGGT-3 ', downstream primer 5 '-TTTCTTTGGCCCTGTATTGATTATT
AGTGGGTCGC-3 ' (underscore part is the complementary series connected for In-fusion clone).Amplification template is middle interstitial granules
pTS-HA/H9.Agarose gel electrophoresis testing goal band about 19kb, is consistent with intended 19.5kb size.PCR primer is entered
Row reclaims purification, measures its concentration, treats that clone connects.
1.2.3 the connection of recombiant plasmid pTS-HA/H9-IL6 and qualification
Use In-fusion to clone interconnection technique, IL6 genetic fragment good for purification and heat-resistant carriers fragment are cloned
Connect (catenation sequence is ACAGGGCCAAAGAAA and GGGAGACATTCAGAG).Connect product and convert DH5 α competent cell,
Cell after conversion is chosen single bacterium colony and is carried out liquid culture after cultivating 16 hours on LB plate.Bacterium solution is carried out the PCR of IL6 gene
Identify.The bacterium solution being accredited as the positive carries out plasmid extraction and carries out enzyme action qualification.Respectively with BamHI and MluI restriction endonuclease to total length weight
Group plasmid carries out enzyme action qualification, has all obtained restriction enzyme mapping in line.Enzyme action is identified correct recombiant plasmid pTS-
HA/H9-IL6 delivers to order-checking company and carries out sequencing, and result shows that HA/H9 and IL6 gene has been inserted into Avian pneumo-encephalitis virus
Heat-resistant carriers, and all sequences is completely the same with expection, recombiant plasmid pTS-HA/H9-IL6 successfully constructs.
Second step, recombinant virus rTS-HA/H9-IL6 rescue recovers and identifies
The rescue of 2.1 recombinant virus rTS-HA/H9-IL6 recovers
Use lipofection, the recombiant plasmid pTS-HA/H9-IL6 built and three are expressed NP, P and L respectively
The helper plasmid cotransfection BHK-21 cell (infecting the vaccinia virus expressing t7 rna polymerase in advance) of albumen.After transfection, 72 is little
Time, multigelation harvesting liquid, inoculate 9-11 age in days SPF Embryo Gallus domesticus.Inoculate latter 5 days, gather in the crops chick embryo allantoic liquid, pending new city
Epidemic disease 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-HA/H9-IL6 saves successfully.
PCR amplification and the order-checking of 2.3 recombinant virus HA/H9 genes and IL6 gene
The specificity amplification primer two of application HA/H9 and IL6 gene is right, to recombinant virus rTS-HA/H9-IL6's respectively
HA/H9 and IL6 gene carries out RT-PCR amplification, pcr amplification product carries out Sequence analysis, all amplification and has arrived expection sheet
Section.Specifying information is respectively as follows: a length of 1774bp of H9/HA (SEQ ID No.1), holds from 5 ' to 3 ', contains P gene successively
Part non-coding area sequence (UTR), the gene termination sequence (GE) of P gene, intermediate sequence (IGS), the gene of HA gene initiate
Sequence (GS), the Kozak sequence of HA gene, HA gene order, the dual termination codon of HA gene, the GE sequence of HA gene,
IGS sequence, the GS sequence of M gene and the UTR sequence of M gene；The a length of 918bp of IL6 (SEQ ID No.2), holds from 5 ' to 3 ',
Contain 3 ' end Leader partial sequence, the gene start sequence (GS) of IL6 gene, the Kozak sequence of IL6 gene, IL6 successively
Gene order, the dual termination codon of IL6 gene, the GE sequence of IL6 gene, IG sequence, the GS sequence of NP gene and NP base
The UTR sequence of cause.Consistent with expected sequence 100%.
The expression checking in recombinant virus rTS-HA/H9-IL6 of 3rd step, H9 subtype avian influenza virus HA albumen
The expression of 3.1 indirect immunofluorescence detection HA albumen
Recombinant virus rTS-HA/H9-IL6 and rTS09-C (comparison) is infected BHK-21 cell, 24h with 0.01MOI respectively
Laggard row indirect immunofluorescene assay, one resists for NDV positive chicken serum or H9 subtype avian influenza positive serum.Testing result table
Bright: recombinant virus rTS-HA/H9-IL6 infect intracellular, use NDV positive chicken serum or H9 subtype avian influenza positive blood
Clearly as one anti-time green fluorescence all can be detected, and the cell that rTS09-C strain is infected only detects under NDV positive serum effect
To fluorescence.
The expression of 3.2 Western Blot method detection HA albumen
Recombinant virus rTS-HA/H9-IL6 and rTS09-C (comparison) is infected BHK-21 cell, 24h with 0.01MOI respectively
Rear collection cell pyrolysis liquid, is one anti-to carry out Western Blot detection with H9 subtype avian influenza positive chicken serum.Testing result
Show: an obvious purpose band occurs in 55-70KDa interval, close with intended 61KDa size.Show that HA albumen exists
The intracellular correct expression of recombinant virus infection.
4th step, the Identification of Biological Characteristics of recombinant virus rTS-HA/H9-IL6
The multiplication characteristic of 4.1 recombinant viruses measures
For the growing multiplication situation of heavier papova rTS-HA/H9-IL6 strain Yu parent's rTS09-C strain, by two strain virus
Respectively with 100TCID50Inoculation 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, compared with parent's rTS09-C strain, and the growth of recombinant virus rTS-HA/H9-IL6 strain
Characteristic has declined, and viral titer is about 106.0TCID50/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-HA/H9-
The MDT/MLD of IL6 is more than 120h, shows that recombinant virus maintains the low toxicity characteristic of parent plant, and the insertion of exogenous gene does not change
Become the pathogenic of recombinant virus.
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-HA/H9-IL6 still has hemagglutination activity after 56 DEG C of 150min of resistance to heat treatment.Show restructuring
The heat-resistant quality of virus rTS-HA/H9-IL6 is consistent with parent plant, is heat-resisting strain.(the preservation of this Strain, is deposited in force
Chinese university China typical culture collection center, preserving number is CCTCC NO:V201630).
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>the heat-resisting vaccine strain of recombinant Newcastle disease of coexpression H9 subtype avian influenza virus HA and chicken interleukin-2 6 albumen and preparation
Method
<160>2
Sequence
--------
<213> OrganismName :
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cagctatatt aaggattaag aaaaaatacg ggtagaaagc ttgccaccat ggaaacaata 60
tcactaatag ctatactact agtagtaaca gtaagcaatg cagataaaat ctgcatcggc 120
taccaatcaa caaactccac agaacctgtg gatacgctaa cagaaaacaa tgtccctgtg 180
acacatgcta aagaattgct ccacacagag cacaatggga tgctgtgtgc aacaaatctg 240
ggacatcctc tcattctaaa cacctgtacc attgaaggac tgatctatgg caacccttct 300
cgtgatcagc tgttgggagg aggaaaatgg tcctacatcg tcgaaagacc atcggccgtt 360
aatggaatgt gttaccctgg gaatgtagaa aacctagagg aactaagatc actctttagt 420
tctgctagtt cctaccaaag aattcggatc tttccagaca cgatctggaa tgtgtcttac 480
aatggaacaa gcaaagcatg ttcagattca ttctacagaa gcatgagatg gctgactcaa 540
aagaacaccg cttaccctat tcaagacgcc caatacacaa ataatagagg aaagagcatt 600
cttttcatgt ggggcataaa tcacccaccc accgatactg tacagacaaa tttgtacaca 660
aggaccgaca caacaacaag tgtgacaaca gaggatataa ctagaacctt caaaccaatg 720
atagggccaa ggccccttgt caatggtcag caggggagaa ttgattatta ttggtcggta 780
ttaaaaccag gtcagacatt gcgaataaga tccaatggga atctaattgc tccatggtat 840
ggacacattc ttccaggaga gagccacgga agaatcctga agactgattt aaacagtggt 900
aactgtgtag tgcaatgtca gactgaaaga ggtggcttaa acaccacatt gccgttccac 960
aatgtcagta agtatgcatt tgggaactgc ccaaagtatg ttggagtaaa gagtctcaga 1020
ctggcagttg gtctaagaaa tgtgcctgct agatcaagta gaggactatt tggggctata 1080
gctggtttca tagagggagg ttggtcaggg ttagtcgctg gttggtatgg gttccagcat 1140
tcaaatgatc aaggggtagg tatggctgca gatagagggt caactcaaag ggcaattgac 1200
aaaataacat ccaaagtgaa taatatagtc gataaaatga acaagcagta tgaaattatt 1260
gatcatgaat tcagcgaggt tgaaactaga ctcaatatga tcaataataa gattgatgat 1320
caaatacaag acatatgggc ttataatgca gaattgctag tgctgcttga aaatcagaaa 1380
acactcgatg aacatgatgc aaatgtgaac aatctatata acaaagtgaa gagggcactg 1440
ggttccaatg ccatggaaga cgggaaagga tgttttgagc tataccataa atgtgctgat 1500
cagtgcatgg agacaattcg gaacgggacc tataacagga gaaagtataa agaggaagca 1560
aaactagaaa gacagaaaat agaaggggtc aagctggaat ctgaaggaac ttacaaaatc 1620
ctcaccattt attcgactgt cgcctcatct cttgtgattg caatggggtt tgctgccttc 1680
ttgttctggg ccatgtccaa tggatcttgc agatgcaaca tttgtatata ataataatta 1740
agaaaaaata cgggtagaat cggagtgccc cgat 1774
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SequenceName : 1
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--------
<213> OrganismName :
<400> PreSequenceString :
tcccccagcc ccacaacccc acctgcccag ggcaacatag gtacaatgcg acccactaat 60
aatcaataca gggccaaaga aattagaaaa aagtacgggt agaaagcagc ttgccaccat 120
gaacttcacc gagggctgcg aggcgacggg acggcggccg gggagcgccg ggagccgccg 180
ccggagagcg ccccgtcccg gccccgtcgc gctgctgccg ctgctgctgc cgctgctgct 240
gccgcccgcc gccgccgtcc cgctgcccgc cgccgcggac tcgtccggag aggttgggct 300
ggaggaggag gcgggggcgc ggcgggcgct gctcgactgc gagccgctgg cccgggtgct 360
gcgcgaccgc gccgtccagc tgcaggacga gatgtgcaag aagttcaccg tgtgcgagaa 420
cagcatggag atgctcgtcc ggaacaacct caacctgccc aaggtgacgg aggaggacgg 480
ctgcctgctc gccggcttcg acgaggagaa atgcctgacg aagctctcca gcggcctgtt 540
cgcctttcag acctacctgg aattcattca agagactttc gatagcgaaa agcagaacgt 600
cgagtctctg tgctacagca caaagcacct ggcggccacg atccggcaga tggtgataaa 660
tcccgatgaa gtggtcatcc cagactcggc cgcccagaaa tccctcctcg ccaatctgaa 720
gtcagataag gactggatag agaaaatcac catgcacctc atcctccgag actttacttc 780
gtttatggag aagaccgtga gggccgttcg ctatttgaaa aagaccagga gtttcagtgc 840
ctaataataa ttaagaaaaa atacgggtag aagggagaca ttcagagatc agggcgagtc 900
acccgggtct ctgctctc 918
<212> Type : DNA
<211> Length : 918
SequenceName : 2
SequenceDescription :
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--------
<213> OrganismName :
<400> PreSequenceString :
cagctatatt aaggattaag aaaaaatacg ggtagaaagc ttgccaccat ggaaacaata 60
tcactaatag c 71
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--------
<213> OrganismName :
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atcggggcac tccgattcta cccgtatttt ttcttaatta ttattatata caaatgttgc 60
atctgc 66
<212> Type : DNA
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SequenceName : 4
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
tcggagtgcc ccgattgtgc caagatggac tc 32
<212> Type : DNA
<211> Length : 32
SequenceName : 5
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
tccttaatat agctgaattg attgcagctg cgcgatc 37
<212> Type : DNA
<211> Length : 37
SequenceName : 6
SequenceDescription :
Sequence
--------
<213> OrganismName :
<400> PreSequenceString :
cagctatatt aagga 15
<212> Type : DNA
<211> Length : 15
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SequenceDescription :
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--------
<213> OrganismName :
<400> PreSequenceString :
tcggagtgcc ccgat 15
<212> Type : DNA
<211> Length : 15
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--------
<213> OrganismName :
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acagggccaa agaaattaga aaaaagtacg ggtagaaagc agcttgccac catgaacttc 60
accgagggct gcga 74
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--------
<213> OrganismName :
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ctctgaatgt ctcccttcta cccgtatttt ttcttaatta ttattaggca ctgaaactcc 60
tggtc 65
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--------
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--------
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--------
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Claims (4)
1. the heat-resisting vaccine strain of recombinant Newcastle disease of coexpression H9 subtype avian influenza virus HA and chicken interleukin-2 6 albumen, its feature exists
It is rTS-HA/H9-IL6 in described heat-resisting vaccine strain Classification And Nomenclature, is preserved in China of Wuhan University allusion quotation on April 19th, 2016
Type culture collection center, preserving number is CCTCC NO:V201630.
2. the preparation side of the heat-resisting vaccine strain of recombinant Newcastle disease of coexpression H9 subtype avian influenza virus HA and chicken interleukin-2 6 albumen
Method, it is characterised in that comprise the steps:
The insertion of 1.1 HA/H9 genes
H9N2 bird flu virus A/Chicken/Hubei/C1/2007 strain is bred on 9-11 day instar chicken embryo, inoculates 5 days
Rear results chick embryo allantoic liquid；The allantoic fluid of results carries out the RT-PCR amplification of HA gene, and amplimer is: forward primer 5 '-
CAGCTATATTAAGGATTAAGAAAAAATACGGGTAGAAAGCTTGCCACCATGGAAAC AATATCACTAATAGC-3 ', under
Trip primer 5 '-ATCGGGGCACTCCGATTCTACCCGTATTTTTTCTTAATTATTATTATATACAAATG TTGCATCTGC-
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 '-
TCGGAGTGCCCCGATTGTGCCAAGATGGACTC-3 ', downstream primer 5 '-
TCCTTAATATAGCTGAATTGATTGCAGCTGCGCGATC-3′；Amplification template is Avian pneumo-encephalitis virus heat-resisting strain plasmid pTS09-
C；Carry out PCR primer reclaiming purification, measure its concentration, treat that clone connects；
The connection of 1.3 recombiant plasmid pTS-HA/H9 and qualification
Use In-fusion to clone interconnection technique, HA genetic fragment good for purification and heat-resistant carriers fragment carried out clone and connects,
Catenation sequence is CAGCTATATTAAGGA and TCGGAGTGCCCCGAT；Connect product and convert DH5 α competent cell, after conversion
Cell cultivate 16 hours on LB plate after choose single bacterium colony and carry out liquid culture；The PCR that bacterium solution carries out HA gene identifies, mirror
The bacterium solution being set to the positive carries out plasmid extraction and carries out enzyme action qualification；
The PCR amplification of 14 IL6 genes
With plasmid pIL6 as template, carrying out the PCR amplification of IL6 gene, amplimer is: forward primer 5 '-
ACAGGGCCAAAGAAATTAGAAAAAAGTACGGGTAGAAAGCAGCTTGCCACCATGA ACTTCACCGAGGGCTGCGA-
3 ', downstream primer 5 '-CTCTGAATGTCTCCCTTCTACCCGTATTTTTTCTTAATTATTATTAGGCACTGAAA CTC
CTGGTC-3；PCR primer carrying out agarose gel reclaim after purification, measure DNA concentration, pending clone connects；
The PCR amplification of 1.5 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 '-
GGGAGACATTCAGAGATCAGGGCGAGTCACCCGGGT-3 ', downstream primer 5 '-
TTTCTTTGGCCCTGTATTGATTATTAGTGGGTCGC-3 ', amplification template is middle interstitial granules pTS-HA/H9, to PCR primer
Carry out reclaiming purification, measure its concentration, treat that clone connects；
The connection of 1.6 recombiant plasmid pTS-HA/H9-IL6 and qualification
Use In-fusion to clone interconnection technique, IL6 genetic fragment good for purification and heat-resistant carriers fragment are carried out clone even
Connecing, catenation sequence is ACAGGGCCAAAGAAA and GGGAGACATTCAGAG；Connect product and convert DH5 α competent cell, convert
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 IL6 gene
Fixed；The bacterium solution being accredited as the positive carries out plasmid extraction and carries out enzyme action qualification；
The rescue of 1.7 recombinant virus rTS-HA/H9-IL6 recovers
Use lipofection, the recombiant plasmid pTS-HA/H9-IL6 built and three are expressed NP, P and L albumen respectively
Helper plasmid cotransfection BHK-21 cell；Transfect latter 72 hours, multigelation harvesting liquid, inoculate 9-11 age in days SPF chicken
Embryo；Inoculate latter 5 days, gather in the crops chick embryo allantoic liquid, isolated coexpression H9 subtype avian influenza virus HA and chicken interleukin-2 6 albumen
The heat-resisting vaccine strain of recombinant Newcastle disease.
3. the recombinant Newcastle disease of coexpression H9 subtype avian influenza virus HA described in claim 1 and chicken interleukin-2 6 albumen is heat-resisting
Vaccine strain application in preparing recombinant virus.
4. the recombinant Newcastle disease of coexpression H9 subtype avian influenza virus HA described in claim 1 and chicken interleukin-2 6 albumen is heat-resisting
Vaccine strain application in preparing H9 bird flu, the heat-resisting live vaccine of newcastle bigeminy.
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| CN201610425143.6A CN105969741A (en) | 2016-06-15 | 2016-06-15 | Recombinant Newcastle disease heat-resisting vaccine strain co-expressing HA and chicken interleukin 6 proteins of H9 subtype avian influenza virus and preparation method thereof |
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| 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 |
| CN107353328A (en) * | 2017-08-23 | 2017-11-17 | 上海市动物疫病预防控制中心 | A kind of H9N2 subtype avian influenza virus sample particles of restructuring and its production and use |
| 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 |
| CN114805537A (en) * | 2022-04-26 | 2022-07-29 | 华南农业大学 | Recombinant plasmid for expressing canine interleukin 6, cell strain for stably expressing canine interleukin 6 protein, and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107353328A (en) * | 2017-08-23 | 2017-11-17 | 上海市动物疫病预防控制中心 | A kind of H9N2 subtype avian influenza virus sample particles of restructuring and its production and use |
| 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 |
| CN114805537A (en) * | 2022-04-26 | 2022-07-29 | 华南农业大学 | Recombinant plasmid for expressing canine interleukin 6, cell strain for stably expressing canine interleukin 6 protein, and preparation method and application thereof |
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