CN110305898A - The rescue of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell - Google Patents
The rescue of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell Download PDFInfo
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Abstract
The present invention relates to animal vaccine technical fields, more particularly to transfect the rescue method of the H9N2 hypotype acclimatization to cold attenuated live vaccine of 293T and DF-1 co-cultured cell.Select one plant of H9N2 hypotype acclimatization to cold attenuated strain TX-25-CE30, 8 plasmids of its gene are expressed in building, with TX-25-CE30Internal gene is skeleton, and using its maternal TX plants of HA, NA gene of strain as external gene, transfection human embryonic kidney cells 293T and chicken fibroblasts system DF-1 co-cultured cell save out the strain weaker to mammalian cell infection ability, be named as rTXca- HA-NA, the recombinant virus are immunized after chicken to the primary immunization protective rate of homologous strain up to 100%.
Description
Technical field
The present invention relates to animal vaccine technical fields, more particularly to the H9N2 hypotype of transfection 293T and DF-1 co-cultured cell
The rescue method of acclimatization to cold attenuated live vaccine.
Background technique
Bird flu is a kind of important epidemic disease for seriously endangering China's poultry husbandry and developing in a healthy way with human health, and serotype is many
More, while carrying out bio-safety, vaccine immunity is still the main means of these epidemic diseases of prevention and control.H9N2 subtype avian influenza exists
Many Asian countries including China and South Korea are popular, are in endemicity in China.The virus infection poultry can be drawn
The clinical symptoms such as egg production reduction, growth retardation are played, occur death often with other diseases mixed infection, to make to aviculture
At sizable economic loss.Since nineteen ninety-eight, China is carrying out always the immunity inoculation meter using oil-emulsion bacterin
It draws.However, in the past twenty years, H9N2 epidemic situation persistently occurs.In the chicken group of China, H9N2 virus at least identifies 4
The different gene group of kind, causes inactivated vaccine immuning failure to happen occasionally due to antigenic variation.Therefore, exploitation reply virus is anti-
The safely and effectively attenuated live vaccine of original drift will be one of the selection method for controlling H9N2 subtype avian influenza.
Compared with traditional inactivated vaccine, attenuated live vaccine can induce body to generate mucous membrane and cellullar immunologic response, tool
There are the advantages such as cross-protection is good, immunization ways facilitate.Attenuated vaccine such as passes on special host there are many weak method is caused
Cause weak, modification Disease-causing gene causes weak, and acclimatization to cold causes weak etc..Wherein acclimatization to cold vaccine has temperature sensitive, and pathogenicity is weaker, returns
The features such as mutation ability is poor again, and genetic stability is good.In the 1960s, the H.F.Massab of University of Michigan expects one
The method for kind influenza virus gene being driven to change: by A/Ann Arbor/6/60 (H2N2) in primary Chicken kidney cell gradient
Cooling passage, it is final to obtain in 25 DEG C of efficient replications, and the mutated viruses of (>=38 DEG C) inefficient duplication at high temperature.With wild type
Virus is compared, and the virus for obtaining acclimatization to cold characteristic shows significantly to cause weak spy in influenza susceptible animal model ferret and mouse
Property, and induction of high-level antibody.In 2003, based on influenza A (the A/Ann Arbor/6/ with acclimatization to cold characteristic
60) nasal spray formula attenuated live vaccine caused by skeleton (in addition to other segments of HA, NA), obtains in the U.S. using approval.
The vaccine is produced by SPF chicken embryo, 2-49 years old crowd can be protected well from the infection of influenza virus.Currently, sharing 5 streams
Influenza Virus acclimatization to cold strain is approved for preparing the donor strain of attenuated live vaccine: i.e. influenza A strain A/Leningrad/134/
17/57 (H2N2), A/Leningrad/134/47/57 (H2N2) and A/Ann Arbor/6/60 (H2N2) and Type B influenza virus
Strain B/USSR/60/69 and B/Ann Arbor/1/66.
Since influenza A infects a variety of species hosts, in addition to being important, human pathogen is external or many poultrys for it
The important pathogen body of fowl such as chicken and pig.Since live vaccine has the advantages that generate cell, body fluid and mucosa-immune, and it is convenient big
Scale is immune, and the live cold-adapted vaccine research and development for aviculture have gradually become a hot spot.
Lee etc. develops H9N2 hypotype acclimatization to cold attenuation by adapting to virus in chicken embryo under the conditions of 25 DEG C
Live vaccine.When acclimatization to cold A/Chicken/Korea/S1/03 influenza virus is inoculated with laying hen, virus can detect in tracheae, and
It is not to be detected in lung, compared with wild type H9N2 influenza infection, does not observe egg production decline and dead generation, expression
The CD8 of IFN-γ+T lymphocyte is induced.When sick with acclimatization to cold attenuation H9N2 A/Chicken/Korea/S1/03 influenza
After poison is immune, when carrying out attacking poison with wild type H9N2 A/Chicken/Korea/S21/04 influenza virus, vaccine protects chicken
Egg production reduction and death do not occur for group.
Wei etc. by chicken embryo continuous gradient cooling passage cultivated H9N2 hypotype acclimatization to cold influenza vaccines Candidate Strain
SD/01/10-ca.The Candidate Strain in chicken body be safe and the chicken in intranasal immunisations in induction of significant blood clotting inhibit anti-
Body level and influenza virus specific C D4+And CD8+T cell immune response.The Candidate Strain can make chicken from homologous and heterologous
The attack of H9N2 virus.
Yang Da etc. by chicken embryo continuous gradient cooling passage obtain H9N2 hypotype acclimatization to cold influenza vaccines Candidate Strain
TX-25-CE30.The virus has had acclimatization to cold characteristic and temperature sensitivity, to the pathogenic significant decrease of mouse.The Candidate Strain is only
Can be in chicken larynx tracheae portion Low-level Replication, and there is no transmission capacity.Primary immunization is capable of providing 60% or more immune protective rate,
80% or more immune protective rate is capable of providing after booster immunization.
These results of study show that acclimatization to cold attenuation H9N2 vaccine can be used for that chicken group is protected to resist H9N2 influenza virus sense
Dye.
Attenuated donor strain is obtained, is the first step of attenuated live vaccine of studying flu virus, it is how that the attenuation of donor strain is special
It is a very crucial step in property " grafting " to vaccine strain.Influenza virus Reverse Genetics, also known as " virus rescue ", are roots
According to the operating system that viral genome and duplication feature are established, the cDNA of virus is mainly obtained using molecule clone technology,
Manual operation is carried out to the genome of virus, obtains the gene composition virus that people want.Hoffmann establishes 8 plasmid influenzas
Virus rescue system becomes the primary turning breakthrough in influenza virus reverse genetics manipulation technology development history.This is one double
To double expression system, i.e., the transcription of viral RNA and the expression of virus protein are realized at the same template (on PHW2000 carrier).
He transfects 293T and MDCK co-cultured cell by constructing minimum plasmid number bidirectional transcription expression vector, greatly improves rescue
Efficiency.The rapid development of Reverse Genetics, the research and development for attenuated influenza virus live vaccine provide unprecedented opportunities.
From 2008, the Medimmune Inc. in the U.S. starts with Reverse Genetics to produce the Seasonal Influenza Vaccine of acclimatization to cold.
Its basic operation is that 8 genetic fragments of influenza virus are cloned into respectively in 8 plasmids, and cotransfection mammalian cell obtains
Obtain active influenza virus particles.Wherein donor strain of 6 internal genes from acclimatization to cold, 2 surface antigen genes HA, NA
From epidemic strain, the in this way Cold tolerance of the existing donor strain of influenza virus of rescue acquisition, and the antigenicity with epidemic strain.Instead
To genetic technique can infected by influenza gene carry out manual operation, be it is external obtain people needed for the influenza virus weight that forms of gene
The powerful tool of group strain.Compared with classical genetic resortment method, this method has the advantages that without screening, quickly to prepare.Mesh
Before, reverse Genetics Technique has become the hot technology of influenza virus research field.
The type and vigor for transfecting cell are to influence one of the key factor of virus rescue efficiency.Select the eukaryon of transfection
Cell, transfection efficiency and purpose virus on cell duplication and growth ability be to be considered an important factor for.Hoffmann
The transfection cell 293T used with very high transfection efficiency and into the cell with enough starting pHW2000 expression at
Point, there is enough rna plymerase is especially in cell；Each hypotype of co-cultured cell MDCK infected by influenza is all quicker
Sense, can produce higher virus titer, which is widely used in the monitoring of influenza.Liu Yanyun, Fan Dandan, Pu Juan
Et al. save out the strain that 8 segments both are from H9N2 hypotype with 293T and MDCK co-cultured cell.Xiao Yuerui with 293T and
The co-cultivation of Vero cell saves out H1N1 subtype avian influenza virus and provides internal gene, and H9N2 hypotype HA, NA gene is external base
The recombination H9N2 virus of cause.Shi Huoying COS-1 cellular rescue goes out full genome from A/Chicken/Shanghai/F/98
(H9N2) strain.The 293T cellular rescue such as Jung goes out recombinant strain rgX-31ca of 8 segments from acclimatization to cold strain X-31ca,
Recombination poison has identical acclimatization to cold characteristic and temperature sensitivity with parent's acclimatization to cold strain.
8 segments can be saved out by pHW2000 expression vector cotransfection 293T and MDCK co-cultured cell both to be from
The strain of H9N2 hypotype, and some avian influenza virus acclimatization to cold strains weak in mammalian cell replication capacity are in 293T and MDCK
It is difficult to save successfully in co-cultured cell.The Cold tolerance for the existing donor strain of influenza virus that rescue obtains is constructed, and is had
How the antigenicity of epidemic strain, the i.e. recombinant virus of " 6+2 " mode improve nonmammalian cells adaptability H9N2 hypotype fowl stream
The rescue efficiency of Influenza Virus is a urgent problem.
Summary of the invention
To achieve the purpose of the present invention, the technical solution adopted by the present invention is that:
The rescue method of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell, comprising:
Viral allantoic fluid total serum IgE is extracted, then expands 8 genetic fragments of virus respectively with reverse transcription PCR；PCR product
After agarose gel electrophoresis, gel purification kit are purified and recycled, it is connect with 3 carrier of pEASY-Blunt, as centre
Transition plasmid；Plasmid carries out digestion after sequence verification sequence is correct, with corresponding endonuclease；The purpose product of digestion passes through
Agarose gel electrophoresis, gel purification kit are cloned into pHW2000 carrier after purification, extract plasmid and are stored in -70 DEG C of rings
Under border.
Further, 8 genetic fragments are respectively as follows: PB2, PB1, PA, HA, NP, NA, M and NS.
Further, amplimer is respectively as follows: Ba-PB2-Fb、Ba-PB2-R、Bm-PB1-Fc、Bm-PB1-R、Bm-PA-F、
Bm-PA-R、Bm-HA-F、Bm-HA-R、Ba-NP-F、Ba-NP-R、Ba-NA-F、Ba-NA-R、Bm-M-F、Bm-M-R、Bm-NS-F
And Bm-NS-R, sequence is respectively as shown in SEQ ID NO.1-16.
It is a further object of the present invention to provide a kind of H9N2 subtype avian influenza virus methods of viral rescue, which is characterized in that
It is transfected using plasmid obtained by the above method.
Further, it is transfected using 293T and DF-1 co-cultured cell.
Further, TPCK pancreatin, the final concentration of 2 μ g/mL of TPCK pancreatin are used in transfection process.
It is a further object of the present invention to provide purposes of the above method in the rescue of H9N2 subtype avian influenza virus.
It is a further object of the present invention to provide the vaccines of above method preparation.
The present invention measure acclimatization to cold strain related biological characteristic, find be suitable for acclimatization to cold strain duplication can passage cell, use
In co-culturing Revive virus with 293T cell, the correlated condition of optimization acclimatization to cold strain duplicating efficiency is explored, for improving rescue effect
Rate.The one plant of H9N2 hypotype acclimatization to cold attenuated strain TX-25-CE for selecting this laboratory culture and saving30, its internal base of building expression
6 plasmids of cause, using its maternal TX plant HA, NA gene of strain as external gene, 8 plasmid co-transfection human embryonic kidney cells 293T and
Chicken fibroblasts system DF-1 co-cultured cell is saved out weaker to mammalian cell (such as 293T, MDCK) infection ability
Strain is named as rTX ca-HA-NA.
Compared with the existing technology, the application achieve it is following the utility model has the advantages that
Cell used in common rescue influenza A is the mammalian-types cell such as 293T, Vero, COS-1, but
Many fowl source stream Influenza Virus show the weaker feature of the infection ability on mammalian cell compared to source of people influenza virus.
During virus rescue, the vRNA and mRNA that are generated by plasmid template transfection mammalian cell and then the viral number being packaged into
Amount is limited, it is therefore necessary to which the replicanism of dependent cells generates more progeny virus.DF-1 as a kind of fowl source cell, compared with
It easily by fowl source influenza infection, is co-cultured with high transfection efficiency cell 293T, can increase and be packed out from 293T cell
Virus particle infection efficiency, to improve the rescue success rate of virus.One plant of H9N2 hypotype acclimatization to cold has successfully been saved in this research
Attenuated strain rTX ca-HA-NA lays the foundation to be subsequent around vaccine candidate strain expansion research.
Detailed description of the invention
Fig. 1 is the growth curve of parental virus and acclimatization to cold strain on 293T cell；
Fig. 2 is the growth curve of parental virus and acclimatization to cold strain on mdck cell；
Fig. 3 is the growth curve of parental virus and acclimatization to cold strain on DF-1 cell；
Fig. 4 is 8 segment positive plasmid PCR amplification result (M:1Kb Plus DNA Ladder；1:PB2；2:PB1；3:PA；
4:HA；5:NP；6:NA；7:M；8:NS)；
Specific embodiment
Embodiment 1
1. acclimatization to cold attenuated strain TCID on different cells50Measurement
Inoculation the previous day spreads cell 293T, MDCK, Vero, COS-1, CEF, DF-1 to be measured to 96 porocyte culture plates
In, single layer is formed to cell, culture supernatant is discarded and is washed 2 times with sterile PBS, final concentration of 2 μ g/mL TPCK pancreas will be added
The Opti-MEM of enzyme subtracts blood serum medium, is seeded in culture plate after 10 times of serial dilution virus liquids, and each dilution connects 4 holes,
Every hole 0.1mL, infected cell is at 33 DEG C, 5%CO2Under the conditions of continue to cultivate, statistics infects positive hole count after 48h, according to
Reed-Muench method calculates virus TCID50.(it is shown in Table 1) as the result is shown, canine kidney cells MDCK is that common influenza virus is susceptible thin
Born of the same parents, TX strain lose the ability effectively replicated on MDCK after the acclimatization to cold passage by chicken embryo, and in common Revive virus
It is relatively low with the replication capacity on the higher human embryonic kidney cells 293T of transfection efficiency, in other common Revive virus transfection cell
Replication capacity on Vero, COS-1 is poor.Replication capacity of the acclimatization to cold strain on CEF is preferable, in the chicken that can be passed at fiber
Also there is certain replication capacity on cell line DF-1.
The maternal virus of table 1 and acclimatization to cold strain TCID in different cells50Measurement
2. the measurement of acclimatization to cold attenuated strain growth curve on different cells
293T, MDCK, DF-1 cell are spread into 35mm dish, after cell forms single layer, with MOI=0.001 dosage
Virus infected cell, after 33 DEG C of absorption 1h, PBS is washed 3 times, remove unadsorbed virion, and add 3mL contain it is dense eventually
Degree is the Opti-MEM culture medium of 2 μ g/mL TPCK pancreatin, and cell continues to cultivate under the conditions of 5%CO2 at 33 DEG C.It is every after infection
It takes Supernatant samples primary every 12h, continues to that 72h, sample are stored in -70 DEG C of refrigerators for use.All samples are used after sampling
CEF cell carries out virus load titration, and Reed-Muench method calculates the TCID of each sample50Value, and draw growth curve.Knot
Fruit shows that (see Fig. 1,2,3), acclimatization to cold attenuated strain can not be in 293T, mdck cell normal replication, can be normally multiple on DF-1
System.
3. the influence of temperature and TPCK pancreas enzyme concentration to acclimatization to cold strain duplicating efficiency
Inoculation the previous day spreads cell 293T, DF-1 to be measured into 96 porocyte culture plates, forms single layer to cell, discards
Culture supernatant is simultaneously washed 2 times with sterile PBS, and the Opti-MEM that final concentration of 1 μ g/mL or 2 μ g/mL TPCK pancreatin is added is subtracted
Blood serum medium is seeded in culture plate after 10 times of serial dilution virus liquids, and each dilution connects 4 holes, every hole 0.1mL, infection
Cell continues to cultivate under the conditions of 5%CO2 respectively at 33 DEG C, 37 DEG C afterwards, and statistics infects positive hole count after infecting 48h, according to
Reed-Muench method calculates virus TCID50.
H9N2 hypotype strain is weak poison, and the ability of infection cell is weaker, it is therefore necessary to the TPCK pancreatin side outside dependent cells
The HA0 of virus is helped to be cracked into HA1 and HA2 to infection cell, but the use of TPCK pancreas enzyme concentration has a certain range, excessively high meeting
Damaging cells, it is too low and effectively viral hemagglutinin cannot be helped to crack.Acclimatization to cold strain is 33 in temperature as shown in Table 2
DEG C, when TPCK pancreas enzyme concentration is 2 μ g/mL, the duplicating efficiency highest on 293T, DF-1.
Viral duplication under 2 different temperatures of table and TPCK pancreas enzyme concentration
4. the building of reverse genetic plasmid
Trizol method extracts viral allantoic fluid total serum IgE, then expands 8 of virus respectively with reverse transcription PCR (RT-PCR)
Genetic fragment.Amplimer is slightly modified, specific primer is shown in Table 3 referring to the research of Hoffmann etc..PCR product is through agarose
After gel electrophoresis, gel purification kit are purified and recycled, it is connect with 3 carrier of pEASY-Blunt, as middle transition plasmid.
Plasmid carries out digestion after sequence verification sequence is correct, with corresponding endonuclease.The purpose product of digestion is solidifying through agarose
Gel electrophoresis, gel purification kit are cloned into pHW2000 carrier after purification, extract plasmid and are stored under -70 DEG C of environment, complete
Fig. 4 is shown in the building of viral genome transcription/expression plasmid, positive plasmid PCR identification.
The viral whole genome amplification primer of table 3
aThe restriction endonuclease sites introduced in primer mark with italic and underscore
bBa is that restriction enzyme BsaI writes a Chinese character in simplified form
cBm is that restriction enzyme BsmBI writes a Chinese character in simplified form
5. transfection
The day before transfection will be spread after 293T and mdck cell and 293T and DF-1 cell mixed in equal amounts into 35mm dish, mistake
Night culture, is transfected when cell area coverage is up to 85% or more.Culture medium is sucked before transfection, is cleaned 2 times with PBS
Afterwards, every hole 1.5mL Opti-MEM culture medium is added.Transfection procedure is referring to LipofectamineTM3000 transfection reagent explanations
Book, includes transcription/expression plasmid (300ng/ plasmid) of 8 segments in rotaring redyeing system, and transfection reagent dosage is 5 μ L/ systems.It sets
After 33 DEG C of carbon dioxide incubator culture 8h, TPCK pancreatin is added to final concentration of 2 μ g/mL.With 8 segments of maternal strain TX
Transfection is positive control simultaneously, plasmid dish is not added as negative control.After 72h, blows off cell and collect supernatant, be inoculated with 9
Embryo is placed in 33 DEG C of incubators by age SPF chicken embryo, 0.4mL/ embryo, and the hemagglutinative titer of embryo is measured after 72h, and potency is positive allantois
Liquid extracts genome after collecting and carries out sequence verification.The result shows that the 8 fragment expression plasmid transfection 293T of maternal strain TX and
The available virus for having hemagglutinative titer when MDCK co-cultured cell, and 6 containing acclimatization to cold internal gene fragments and 2 TX female parents
The expression plasmid of strain HA and NA segment does not obtain the virus of hemagglutinative titer when transfecting 293T and MDCK co-cultured cell；Repeatedly
Repetition still fails.The 8 fragment expression plasmid transfections of maternal strain TX and 6 internal gene fragments containing acclimatization to cold and 2 TX
Maternal strain HA and the expression plasmid of NA segment obtain the disease of hemagglutinative titer when transfecting 293T and DF-1 co-cultured cell
Poison；Sequencing result shows that purpose strain is saved successfully.Strain and corresponding HA-HI test are as shown in table 4.
Table 4 saves malicious HA-HI test
6. saving the mitotic stability measurement of strain
It will the four anti-PBS dilutions 10 of rescue poison410 age in days SPF chicken embryos of inoculation passage again, the virus that harvest is continuous passage 5 times
Liquid freezes in -70 DEG C of refrigerators, and per generation strain HA-HI test is as shown in table 5.Extract in the 5th generation allantoic fluid viral RNA, reverse transcription simultaneously
Expand 8 fragment genes.Company is sent to be sequenced pcr amplification product to confirm the fidelity for passing on 5 virus gene sequences through chicken embryo
Property.Viral rTXca- HA-NA can stablize passage, carry out sequencing to the 5th generation virus, sequencing result is correct.
5 recombinant virus of table passes on HA potency
7. saving strain acclimatization to cold characteristic, temperature sensitivity identification
After the recombinant virus allantoic fluid that passage is completed dilutes 1000 times, inoculated into chick embryo, 0.2mL/ embryo is placed in difference two-by-two
Temperature (26 DEG C, 33 DEG C, 41 DEG C) under cultivate, after 72h by chicken embryo be placed in 4 DEG C overnight, measure HA-HI tests.The strain of rescue
rTXca- HA-NA has apparent acclimatization to cold characteristic and temperature sensitivity.As a result as follows:
Duplication titre of the poison in chicken embryo is recombinated under 6 different temperatures of table
8. saving the measurement of strain Basic Biological Character
Chicken embryo median infective dose (50%egg infectious dose, EID50): viral allantoic fluid does continuous 10 with PBS
It dilutes again, takes 6 dilutions (10-4-10-9) 10 age in days SPF chicken embryos of inoculation, 4 embryos of each dilution inoculation, 0.1mL/ embryo.It connects
The chicken embryo of kind is cultivated under the conditions of 33 DEG C, and every 12h is primary according to embryo, until 72h, finally calculates according to Reed-Muench method
EID50。
Tissue culture median infective dose (50%tissue culture infectious dose, TCID50): 9-10 days
The SPF chicken embryo in age makes CEF cell, with trypsin digestion and counts after cell culture overnight, is laid on 96 porocyte culture plates
In, it after cell forms fine and close single layer, discards culture supernatant and is washed 3 times with sterile PBS, 2 μ g/mL TPCK pancreatin will be contained
Poison disease vaccination of the nonreactive without blood M199 10 times of serial dilutions of culture medium into culture plate, each dilution connects 4 holes, every hole
0.1mL, infected cell is at 33 DEG C, 5%CO2Under the conditions of continue to cultivate, statistics infects positive hole count after infecting 72h, according to
Reed-Muench method calculates virus TCID50.As shown in table 7, recombination acclimatization to cold strain has preferable infection chicken embryo and chick embryo fibroblast
The ability of cell.
Table 7 recombinates the Basic Biological Character of poison
9. protest test
With 106EID50The virus quantity of/0.1mL is immunized 3 week old SPF chickens, at the same using the chicken of only inoculation sterilizing PBS as pair
According to group.After immune 21d, with 106EID50The TX of/0.1mL dosage attacks poison by collunarium eyedripping way.Attack the 3rd, 5,7 natural gift after poison
Not Cai Ji throat swab and cloacal swabs to measure toxin expelling situation.As the result is shown for the poison of attacking of maternal TX, rTXca-HA-NA
Immune group equal not toxin expelling at 3,5,7 days, can provide 100% protection (table 8).
8 SPF chicken vaccine candidate strain Immunoprotection test of table
aOropharyngeal swab, throat swab；bCloacal swab, cloacal swab
In conclusion research shows that non-lactation can be saved out by transfecting 293T and DF-1 cell by " 6+2 " expression plasmid
Zooblast adaptability H9N2 subtype avian influenza virus acclimatization to cold strain, obtain virus have as acclimatization to cold strain acclimatization to cold and
Temperature sensitivity can attack 100% protection of poison offer after chicken is immunized to homologous wild strain, can be used as prevention and control H9N2 subtype avian influenza
Vaccine candidate strain.
Sequence table
<110>Yangzhou University
<120>rescue of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tattggtctc agggagcgaa agcaggtc 28
<210> 2
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atatggtctc gtattagtag aaacaaggtc gttt 34
<210> 3
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tattcgtctc agggagcgaa agcaggca 28
<210> 4
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atatcgtctc gtattagtag aaacaaggca ttt 33
<210> 5
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tattcgtctc agggagcgaa agcaggtac 29
<210> 6
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
atatcgtctc gtattagtag aaacaaggta ctt 33
<210> 7
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tattcgtctc agggagcraa agcagggg 28
<210> 8
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atatcgtctc gtattagtag aaacaagggt gtttt 35
<210> 9
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
tattggtctc agggagcaaa agcagggtag at 32
<210> 10
<211> 34
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atatggtctc gtattagtag aaacaagggt attt 34
<210> 11
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tattggtctc agggagcaaa agcaggagt 29
<210> 12
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
atatggtctc gtattagtag aaacaaggag tttttt 36
<210> 13
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
tattcgtctc agggagcaaa agcaggtag 29
<210> 14
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
atatcgtctc gtattagtag aaacaaggta gttttt 36
<210> 15
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
tattcgtctc agggagcaaa agcagggtga c 31
<210> 16
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
atatcgtctc gtattagtag aaacaagggt gtttt 35
Claims (8)
1. the rescue method of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell, comprising:
Viral allantoic fluid total serum IgE is extracted, then expands 8 genetic fragments of virus respectively with reverse transcription PCR；PCR product is through fine jade
It after sepharose electrophoresis, gel purification kit are purified and recycled, is connect with pEASY-Blunt3 carrier, as middle transition matter
Grain；Plasmid carries out digestion after sequence verification sequence is correct, with corresponding endonuclease；The purpose product of digestion is through agarose
Gel electrophoresis, gel purification kit are cloned into pHW2000 carrier after purification, extract plasmid and are stored under -70 DEG C of environment.
2. the rescue side of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell according to claim 1
Method, which is characterized in that 8 genetic fragments are respectively as follows: PB2, PB1, PA, HA, NP, NA, M and NS.
3. the rescue side of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell according to claim 1
Method, which is characterized in that amplimer is respectively as follows: Ba-PB2-Fb、Ba-PB2-R、Bm-PB1-Fc、Bm-PB1-R、Bm-PA-F、Bm-
PA-R, Bm-HA-F, Bm-HA-R, Ba-NP-F, Ba-NP-R, Ba-NA-F, Ba-NA-R, Bm-M-F, Bm-M-R, Bm-NS-F and
Bm-NS-R, sequence is respectively as shown in SEQ ID NO.1-16.
4. a kind of H9N2 subtype avian influenza virus saves method, which is characterized in that use the described in any item sides of claim 1-3
The plasmid that method obtains is transfected.
5. H9N2 subtype avian influenza virus according to claim 4 saves method, which is characterized in that use 293T and DF-1
Co-cultured cell is transfected.
6. H9N2 subtype avian influenza virus according to claim 5 saves method, which is characterized in that used in transfection process
TPCK pancreatin, the final concentration of 2 μ g/mL of TPCK pancreatin.
7. purposes of the method for any of claims 1-3 in the rescue of H9N2 subtype avian influenza virus.
8. the rescue side of the non-susceptible H9N2 hypotype acclimatization to cold avian influenza virus of mammalian cell according to claim 1 to 3
The vaccine of method preparation.
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| 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 |
| CN114836390A (en) * | 2022-06-13 | 2022-08-02 | 华南农业大学 | H9N2 subtype avian influenza virus MDCK cell cold-adaptation attenuated live vaccine strain and cultivation method and application thereof |
| CN114836390B (en) * | 2022-06-13 | 2023-05-02 | 华南农业大学 | H9N2 subtype avian influenza virus MDCK cell cold-adaptation attenuated live vaccine strain and application thereof |
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