CN104073470A - Spinner-flask culture method for H9N2 subtype of avian influenza virus - Google Patents
Spinner-flask culture method for H9N2 subtype of avian influenza virus Download PDFInfo
- Publication number
- CN104073470A CN104073470A CN201410306875.4A CN201410306875A CN104073470A CN 104073470 A CN104073470 A CN 104073470A CN 201410306875 A CN201410306875 A CN 201410306875A CN 104073470 A CN104073470 A CN 104073470A
- Authority
- CN
- China
- Prior art keywords
- cell
- influenza virus
- virus
- avian influenza
- mdck
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses a spinner-flask culture method for H9N2 subtype of avian influenza virus. The spinner-flask culture method comprises a step of culturing MDCK (Madin-Darby canine kidney)-HA cells, namely culturing the MDCK-HA cells in a spinner flask; a step of inoculating the MDCK-HA cells with the H9N2 subtype of avian influenza virus; particularly, in the inoculating step, when monolayer cells in the cells growing in the spinner flask are more than 90%, the H9N2 subtype of avian influenza virus is inoculated and incubated for 1-2 hours in a cell incubator, and 50ml of 2% NCS (new-born calf serum) DMEM (Dulbecco modified eagle medium) maintenance solution is added after the incubation is ended, and the cells inoculated with the H9N2 subtype of avian influenza virus are cultured in the cell incubator, and finally cell supernatant is collected. According to the spinner-flask culture method disclosed by the invention, the MDCK-HA cell system is adopted, and the conditions of spinner-flask culture are optimized, thus the H9N2 subtype of avian influenza virus is rapidly proliferated to reach a high virus titer and obtain a good immune effect.
Description
Technical field:
The invention belongs to bioengineering field, relate in particular to a kind of cultural method of cell, is a kind of spinner culture method of H9N2 subtype avian influenza virus specifically.
Background technology:
The bird height contagious disease that bird flu (Avian Influenza, the AI) A of Shi You orthomyxoviridae family type influenza virus (Avian Influenza Virus, AIV) causes, has 16 HA hypotypes and 9 NA hypotypes.The difference pathogenic according to avian influenza virus, can be divided into Highly Pathogenic Avian Influenza Virus (HPAIV) and Lowly Pathogenic Avian Influenza Virus.At present, the low pathogenicity bird flu epidemic situation of China mainly be take H9 hypotype as main.China is since reported first chicken group in 1994 is separated to H9N2 hypotype AIV, and this subtype avian influenza virus is extensively present in the most areas such as China Liaoning, Anhui, Shandong, Guangdong, Fujian, Jiangsu, Henan, Hunan, Hubei, Shanghai, Guangxi, Yunnan, Sichuan, Xinjiang.Although H9N2 subtype avian influenza virus does not cause the bird mortality of infection, but because this virus disseminating is very competent, existence range is extremely wide, cause egg drop reduction, immunosuppressive disease, often cause high mortality with other cause of disease coinfection, caused huge financial loss to China's aviculture.Further research is found, H9N2 hypotype AIV can propagate alternately between different hosts chicken, duck, quail, pigeon, has further promoted the popular of this virus.Meanwhile, all right infected pigs of H9N2 hypotype AIV, people's (data of announcing according to CDC 1999,2003, infection event all occur for 2007 in Hong Kong) etc.Therefore, H9N2 subtype avian influenza virus presents more and more consequence on Economic development and public health security, also more and more causes people's extensive concern.
Vaccination is that birds flu-preventing occurs and propagates the most effective means, to improving the poultry disease prevention and control level of China, to ensureing that the sound development of China's aviculture has very positive meaning.At present, the H9N2 subtype avian influenza vaccine of China is chick embryo allantoic liquid inactivated vaccine, and these H9N2 avian influenza virus deactivation vaccine vaccine strains were all before 2002 separated obtain.Research discovery, the strain antigenicity between 1998-2006 is unchanged, and the protection ratio of vaccine is 100%.But after 2006, under the selective pressure of vaccine, between the fowl influenza virus strain of H9N2 hypotype, wide variation have occurred antigenicity, the protection ratio of vaccine is constantly declining.The portion Surveillance demonstration in 2009 of Shanghai Municipal Center for Animal Disease Control & Prevention, the sample H9 hypotype AIV average positive rate in three large live-bird wholesale markets, Shanghai is 8.14%.Wherein in immunity H9N2 hypotype AIV deactivation vaccine and Antibody qualification rate be all greater than in 70% sample and be separated to 45 strain H9 hypotype AIV viruses (45/195), in the Antibody qualification rate of immune H9N2 hypotype AIV deactivation vaccine reaches 100% sample, be separated to 8 strain H9 hypotype AIV (8/90).These results show, after the immunity of existing commercialization H9 hypotype inactivated vaccine, chicken body produces higher immune antibody, but still can not stop virus replication completely, effectively protects.Thereby, the commercially available vaccine that market demand is prepared with existing H9N2 subtype avian influenza virus epidemic strain.
At present, chicken embryo is the main raw that avian influenza virus vaccine is produced and studied, PR8 virus is that a strain chicken embryo adapts to virus strain, also be one of chicken embryo high yield strain at present, in avian influenza vaccine development, usually by HA and the NA gene recombination (6+2) of 6 internal gene of PR8 and epidemic isolates, and recombinant virus is improved to virus titer as vaccine strain.Yet chicken embryo culture influenza virus vaccine has serious defect, when epidemic situation occurs, cannot provide enough chicken embryos, thereby cannot expand in a short time vaccine product.Virus amplification can be subject to the impact of maternal antibody in chicken embryo, makes virus titer can not reach the needs of production of vaccine; Chicken embryo culture virus produces a large amount of refuses, and these treatments of wastes produced not only need to consume a large amount of energy, and easily cause environmental pollution.The influenza virus of cell cultures, can overcome these shortcomings, thereby becomes one of main direction of current influenza vaccines development work.The amplification of inactivated influenza virus seedling seed culture of viruses at leisure by chicken embryo to cell transformation, and more and more demonstrate economic benefit and social benefit.Human influenza virus has obtained important breakthrough in the research of cell vaccine, the world such as Baxter and Novartis vaccine major company, with microcarrier cell large scale culturing technical research success people, use influenza vaccines, and ratify listing by European Union, produced huge economic benefit.Aspect animal influenza virus, because the susceptibility of cell infected by influenza is not as good as chicken embryo, thereby the prepared avian influenza virus titre of cell is not ideal enough, further aggravated the economic constraints of fowl with vaccine, up to the present not yet there is the cell vaccine launch of avian influenza virus.
At present, the contriver's H9N2 of strain more than 20 subtype avian influenza virus that different areas are separated to from 2008-2009 2 years has carried out sequential analysis and antigenicity analysis, screen the strain that a strain antigenicity is good [A/Chicken/Shanghai/441/2009 (H9N2) is called for short SH441].Preliminary cross protection experiment demonstration, vaccine prepared by this strain can be protected in the past and the attack of nearest epidemic isolates preferably.Utilize influenza virus reverse genetic manipulation technology, obtained recombinant virus [referred to as SH441/PR8 (NS2 E67 & 74S) the virus] (Xu great Wei that PR8 is skeleton of take that contains SH441HA, NA and PR8 virus N S2 amino acid mutation, the Preliminary development > > of Ph D dissertation < < H9N2 subtype avian influenza virus in 2012 and the research of duck tembusu virus biological property and vaccine, http://www.doc88.com/p-7864338622047.html).The growth performance of this recombinant virus on cell is better than the malicious PR8 of parent.Except virus transformation, we are also optimized cultured cells, by the HA gene of H5 hypotype AIV being inserted to the genome of mdck cell, set up MDCK-HA clone (patent No. ZL201010106774.4).Due to the H5HA albumen of expressing on cytolemma, make the HA0 albumen of the filial generation influenza virus of this clone MDCK-HA propagation generation, the protease cracking that can use host is HA1 and HA2 (being that influenza virus has infective prerequisite).Thereby this clone can be supported low pathogenicity influenza virus image height virulence influenza virus (as H5 hypotype) fast breeding on cell well, thereby reaches higher virus titer.Under these bases, we to H9N2 subtype avian influenza virus particularly [SH441/PR8 (NS2 E67 & 74S)] on rolling bottle, preparation method is optimized and applies.
Summary of the invention
The object of the present invention is to provide a kind of spinner culture method of H9N2 subtype avian influenza virus, the spinner culture method of described this H9N2 subtype avian influenza virus will solve fowl in prior art and use the virus titer in cell vaccine not high, thereby leads the technical problem of immunogenic poor effect.
The spinner culture method of a kind of H9N2 subtype avian influenza virus of the present invention, comprises the following steps:
1) step of cultivating MDCK-HA cell, is positioned over MDCK-HA cell in rolling bottle, adds nutrient solution, and rolling bottle is positioned over and in cell culture incubator, cultivates 18～22h;
2) step of inoculating H9N2 subtype avian influenza virus in MDCK-HA cell, the cell in rolling bottle grows up to monolayer cell and reaches 90% when above, discards nutrient solution, adopt PBS solution to wash 1～5 time, and discard PBS solution, and inoculation H9N2 subtype avian influenza virus, the dosage of inoculation is 8 * 104TCID50, in cell culture incubator, hatch 1～2 hour, every 10～20 minutes, shake rolling bottle once, hatch after end, discard virus liquid, adopt PBS to wash 1～5 time, then discard PBS;
3) then add 2%NCS DMEM maintenance medium, the volume of described DMEM maintenance medium is 50ml, is placed in cell culture incubator and cultivates, and cultivates 48 hours, collects cell conditioned medium.
Further, after inoculation, every 15 minutes, shake rolling bottle.
Further, described H9N2 subtype avian influenza virus is A/Chicken/Shanghai/441/2009 or A/chicken/Shangdong/A2093/2012.
Further, described H9N2 subtype avian influenza virus is SH441/PR8 (NS2E67 & 74S).
Further, described 2%NCS DMEM maintenance medium represents to add in every 98ML DMEM nutrient solution the calf serum of 2ml again.
The MDCK-HA clone the present invention relates to, its specific name is: the screech owl short-tail sleuth kidney cell line MDCK-HA of stably express Highly Pathogenic Avian Influenza Virus (HPAIV) HA albumen, on January 8th, 2010, be preserved in Chinese Typical Representative culture collection center (Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan, China university, 430072), deposit number is CCTCC NO:C200968.
MDCK-HA clone is to set up by the HA gene of H5 hypotype AIV being inserted to the genome of mdck cell, MDCK-HA clone is due to the H5HA albumen of expressing on cytolemma, the HA0 albumen that makes the filial generation influenza virus of this clone MDCK-HA propagation generation, the protease cracking that can use host is HA1 and HA2 (being that influenza virus has infective prerequisite).Thereby this clone can support low pathogenicity influenza virus as highly pathogenicity influenza virus (as H5 hypotype) fast breeding on cell well, thereby reaches higher virus titer.On these bases, the present invention to H9N2 subtype avian influenza virus particularly [SH441/PR8 (NS2E67 & 74S)] cultural method on rolling bottle be optimized.The present invention finds that the virus infection dosage of selecting is 8 * 10
4tCID
50rolling bottle cell connects after poison, adopt 2%NCS DMEM maintenance medium to cultivate MDCK-HA cell amplification virus, final volume culturing cell by 50ml maintenance medium, receive the poison time for connecing after the rear 48h of poison, H9N2 subtype avian influenza virus is particularly bred well on the MDCK-HA cell of [SH441/PR8 (NS2E67 & 74S)] virus in rolling bottle, and the hemagglutinative titer of cell conditioned medium can reach 2
12, meet the requirement that vaccine candidate strain is tired.
The present invention compares with prior art, and its technical progress is significant.Fowl with cell vaccine owing to carrying out expensive concentrating as people uses, so the virus titer in cell vaccine is not high, thereby the poor effect of immunity causes the market-oriented failure of vaccine for fowl.The present invention has adopted MDCK-HA clone, and the condition in spinner culture process is optimized, can be so that H9N2 subtype avian influenza virus [SH441/PR8 (NS2 E67 & 74S)] fast breeding particularly, thereby reach higher virus titer, improved immune effect.Virus titer is substantially consistent with the titre of chicken embryo, has broken through important research bottleneck, thereby is that good basis has been created in the marketization.
Accompanying drawing explanation
Fig. 1 shows pMX-HA recombinant plasmid structure iron.
Fig. 2 expresses the immunofluorescence figure of the clone of H5HA.Wherein, left figure is the fluorogram of expressing the clone of H5HA, and right figure is control cells.
Fig. 3 shows that MDCK-HA clone Western blot identifies.Wherein, M: albumen dyes Marker in advance; 1: the total protein of normal MDCK; The total protein of 2:MDCK-HA cell.
Fig. 4 shows that PCR detects transcribing of HA in MDCK-HA clone.M:DNAMarker wherein; CDNA first chain of total RNA reverse transcription of 1:MDCK-HA cell is template amplification HA fragment.
Fig. 5 shows H9N2 virus multiplication result.
Fig. 6 A-6D shows that respectively using mdck cell of the present invention is to carry out proliferation of influenza virus to test resulting result.What wherein, Fig. 6 A showed is the propagation result of PR8; What Fig. 6 B showed is the propagation result of H3N1 virus; What Fig. 6 C showed is the propagation result of H4N1; What Fig. 6 D showed is the propagation result of H10N8.
Fig. 7 has shown SH441/PR8 (NS2E67 & 74S) and the growth curve of wild malicious SH441 on MDCK-HA.
Fig. 8 has shown the HI antibody horizontal after immune vaccine.
Embodiment:
1 material
1.1 cells, bacterial classification and plasmid
This tests needed MDCK-HA cell, and 293T cell, JM109 competence, carrier PBD and viral PR8 reverse genetic operating system, H9N2 subtype avian influenza virus [A/chicken/Shanghai/441/2009 (H9N2)] Dou You China Agriculture Academe Shanghai Veterinary Institute aquatic bird influenza laboratory provide.
1.2SPF chicken embryo
Test required SPF chicken embryo all purchased from Beijing Mei Liya company.
1.3 main agents
Liposome 2000, Opti-MEM is purchased from Invitrogen company; DMEM nutrient solution is purchased from GIBCO company; Calf serum, foetal calf serum are purchased from Canadian PAA Laboratories Inc; Pryobest archaeal dna polymerase is purchased from precious biotech firm; TPCK-Trypsin is purchased from Sigma company.
Rescue and the checking of embodiment 1SH441/PR8 (NS2E67 & 74S) mutated viruses
1, the structure of recombinant plasmid PBD-NSE67/74S
PR8 virus strain NS2 transgenation: with (the Chinese veterinary science of reference of the plasmid with PR8NS gene, 2010,40:788)) be template, utilize Pyrobest archaeal dna polymerase (TakaRa), with SapI-NS-F, PR8-NS2-204R and PR8-NS2-193F, SapI-NS-R (table 1), carry out respectively pcr amplification.Two PCR products 1 that obtain and 2, rubber tapping purifying.Above-mentioned two the PCR purified products of take are template, with SapI-NS-F and SapI-NS-R (table 1), carry out pcr amplification, obtain PCR product 3.Use respectively BSPQI (NEB) enzyme to cut the PCR product 3 of purifying and PBD carrier, then both are connected, transform the competent cell in JM109.Picking positive bacteria, extracting plasmid.Through PCR, identify the recombinant plasmid PBD-NS2E67/74S of the doubtful positive, send company to carry out sequence verification.
Sequencing result shows, the NS2 aminoacid sequence on PBD-NSE67/74S plasmid is the 67th and the 74th sudden change that expection has occurred simultaneously only, construction of recombinant plasmid success.Table 1 is saved virus and is identified primer used
2, the structure of recombinant plasmid PBD-441HA, PBD-441NA
By H9N2 subtype avian influenza virus [A/chicken/Shanghai/441/2009 (H9N2)] (laboratory preservation), by Trizol (Invitrogen) extracting RNA, and reverse transcription becomes cDNA the first chain.CDNA the first chain of take is template, uses respectively SapI-HA-F, SapI-HA-R and SapI-NA-F, SapI-NA-R (table 1) to carry out pcr amplification, obtains the PCR purified product of 441-HA and 441-NA, carries out respectively BSPQI enzyme and cuts.Enzyme is cut the PBD carrier that purified product 441-HA and 441-NA cut processing with same enzyme respectively and is connected, and transforms the competent cell in JM109.Picking positive bacteria, extracting plasmid.Through PCR, identify recombinant plasmid PBD-441HA and the PBD-441NA of the doubtful positive, send company to carry out sequence verification.
Sequencing result shows, recombinant plasmid PBD-441HA and PBD-441NA sequence are in full accord with expection, construction of recombinant plasmid success.
3, the preparation of high purity plasmid
With ultrapure extraction agent box (OMEGA), extract the recombinant plasmid that aforesaid method builds: comprise PBD-441HA, PBD-441NA, PBD-NSE67/74S and and the PBD-PR8PB1, the PBD-PR8PB2 that preserve of this laboratory, PBD-PR8PA, PBD-PR8NP, PBD-PR8M (with reference to Chinese veterinary science, 2010,40:788), and measure plasmid concentration.
4, rescue obtains restructuring PR8 mutated viruses
By above-mentioned plasmid, utilize liposome 2000 cotransfections in 293T cell.6h after transfection, discards cell conditioned medium, adds 2ml OPTI-MEM, is placed in the CO of 37 ℃
2in incubator, cultivate 72h.After cell conditioned medium after transfection is processed with TPCK-Trypsin, be inoculated in 9-11 age in days SPF chicken embryo (the logical laboratory animal technology of Beijing Cimmeria dimension company limited), with paraffin sealing, be placed in 37 ℃ of brooders and continue to hatch.After 48-72h, put into 4 ℃ and spend the night, take out, results chick embryo allantoic liquid.Allantoic fluid is measured and is had or not agglutination activity with hemagglutination test.
Result shows, the present invention saves and obtained containing the HA of H9N2 subtype influenza virus and the PR8 of NA gene sudden change recombinant virus, called after SH441/PR8 (NS2E67 & 74S).
5, the evaluation of recombinant virus
With the total RNA of allantoic fluid of Trizol extracting recombinant virus, and with the reverse transcription of 12bp primer, obtain cDNA the first chain.CDNA the first chain of take is template, with SapI-HA-F and SapI HA-R, SapI-NA-F and SapI-NA-R, SapI-NS-F, SapI-NS-R, is upstream and downstream primer, by the method for PCR increase respectively HA, NA and NS fragment, will after these PCR product purifications, check order.
The contained fragment of sequencing result confirmation recombinant virus is all expection, there is no the sudden change outside amount.
Embodiment 2: the mdck cell system that sets up stably express HA albumen
1. construction recombination plasmid pMX-HA
The H5 gene (NCBI accession No NC_007362) of take is template, adds sterilizing distilled water 19.5 μ l, 10 * PCR damping fluid, 2.5 μ l, and dNTP Mix (10mmol/L) 1 μ l, 10umol/L upstream primer (sequence is:
gCGGCCGCaAAATGGAGAAAATAGTGC (SEQ ID NO:1), underscore is partly NotI restriction enzyme site) 1 μ l, 10 μ mol/L downstream primers (sequence is:
cTCGAGtTAAATGCAAATTCTGCATTG (SEQ ID NO:2), underscore is partly XhoI restriction enzyme site) 1 μ l, Taq Plus archaeal dna polymerase (TAKARA) 0.5ul, mixes, and enters PCR reaction.Pcr amplification condition: 94 ℃ of denaturation 5min, 94 ℃ of sex change 15s, 53 ℃ of annealing 15s, 72 ℃ are extended 2min, totally 30 circulations, last 72 ℃ of insulation 10min.
PCR product is after test kit (Axygen company) purifying reclaims, after NotI and xhoI double digestion, be subcloned into the pMX carrier [Onishi of same restrictions endonuclease digestion, M., et al.1996.Exp.Hematol.24:324 – 329], Transformed E .coli JM109 competent cell (TAKARA), coating is dull and stereotyped containing the LB of 100g/ml penbritin, picking resistance bacterium colony enlarged culturing extracting plasmid.Plasmid is cut after evaluation through PCR, enzyme, and serves Hai Boshang Bioisystech Co., Ltd sequence verification.The recombinant plasmid called after pMX-HA (as shown in Figure 1) successfully constructing.
2. transfection 293T cell
To specifications, with test kit (OMEGA), carry out ultrapure plasmid PMDSV (VSVG), MDSV (gag/pol), NF-KB[Zejun Li, et al.JVI, 2009,83 (9): 4153 – 62], pMX-HA extracting carry out electrophoresis detection, finally with Nanodrop2000c ultra-violet and visible spectrophotometer, measure OD260, OD280, calculate DNA content and purity.
By the PMDSV of above-mentioned ultrapurity extracting (VSVG), PMDSV (gag/pol), NF-к B and pMX-HA plasmid, by means of liposome 2000, be transfected in 293T cell.After transfection 6h, discard cell conditioned medium, add OPTI-MEM (Invitrogen), cell is put in the CO of 37 ℃
2in incubator, cultivate.
3. set up the mdck cell system (MDCK-HA clone) of stably express H5HA albumen
293T cell conditioned medium after above-mentioned transfection and mdck cell are contained to 5%CO at 37 ℃
2incubator in hatch cultivation, infect after 24h, discard cell conditioned medium, with PBS, wash twice, then with trysinization, be prepared into individual cells suspension, be inoculated in Tissue Culture Dish, at 5%CO
237 ℃ of incubators in cultivate.Every 3 days, change a not good liquor, add the DMEM nutrient solution containing 5% new-born calf serum (NCS) and 5ug/ml tetracycline.After 15 days, with the little pipeloop of glass, live cell and add trysinization to become individual cells suspension, and sucking-off cell cultures is in 24 porocyte culture plates.After 8 days, continue aforesaid operations clone cell twice.Finally, the clone of having cloned, biography is carried out checking work after ten generations.
The clone that obtained thus, is preserved in Chinese Typical Representative culture collection center (CCTCC, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan, China university, 430072) on January 8th, 2010, and deposit number is CCTCC NO:C200968.
Embodiment 3: the checking of clone
1. indirect immunofluorescence is identified
Until MDCK-HA cell, cover with to 90% time, with PBS, clean cell, dry.Cell, at-20 ℃, with infiltration in the mixed solution of acetone and methyl alcohol and fixing, then cleans with PBS.PBS-M-T for cell (containing 5% skim-milk, 0.05%Tween-20,0.1%Triton X-100), hatches 90min for 37 ℃.With 200 μ L, containing the PBST of 0.25%Tween-20, clean 3 times each 3min.Add respectively H5 polyclonal antibody with PBS (PBS-M-T) dilution containing 5% skim-milk, 0.05%Tween20 (maybe can use the H5 antibody of commercially available acquisition, for example Influenza A Hemagglutinin H5 (Avian H5N1) mAb (
www.antibodies-online.com/terms.htm, Germany), put at 37 ℃ and hatch 1h.Clean the same.Add PBS-M-T to dilute 50 μ L FITC-sheep anti-mouse iggs (KPL, 1:200), incubation 1h at 37 ℃, cleans the same.Finally, with inverted fluorescence microscope (OLYMPUS) observations Taking Pictures recording, specifically referring to Fig. 2.Experimental result finds that HA albumen is expressed on MDCK-HA cytoplasm and cytolemma galore, but fails to be expressed in normal mdck cell.
2.Western blot identifies
Cloning cell line MDCK-HA gets 1-2 * 10
7cell, after ice-cold PBS washing 2 times, add 100 μ l RIPA lysates (1%Triton X-100,1% Sodium desoxycholate, 150mM NaCl, 10mMTris-HCl[pH7.4], 10mM EDTA, 0.1%SDS), ice bath 5min, after the centrifugal 10min of 12,000 * g, collect supernatant.Through carrying out after separation on 12%SDS-PAGE, electrotransfer is to nitrocellulose filter.Nitrocellulose filter after 5% skim-milk sealing, respectively with the anti-H5HA polyclonal antibody of mouse (maybe can use the H5 antibody of commercially available acquisition, for example Influenza A Hemagglutinin H5 (Avian H5N1) mAb (
www.antibodies-online.com/terms.htm, Germany), hatch 1h for 37 ℃, then after PBST washing, hatch 1h with 37 ℃ of the sheep anti-mouse iggs of horseradish peroxidase-labeled, finally by AEC, develop the color.The negative contrast of normal mdck cell total protein is set.As shown in Figure 3, MDCK-HA clone can be expressed H5HA albumen to Western blot result, and in the cracking total protein of the normal full cell of MDCK, fails to detect the expression of H5HA albumen.
3.RT-PCR identifies
3.1 extracted total RNA
Mdck cell and the MDCK-HA cell of cultivating proper amt, add Trizol solution (Invitrogen) cracking, and room temperature is placed 5min; Add 200 μ l chloroforms, firmly shake 15s, room temperature is placed 2-3min; 12,000 * g, 4 ℃, centrifugal 15min; Get upper strata water to new pipe, add 0.5ml Virahol, mix, room temperature is placed 10min; 12,000 * g, 4 ℃, centrifugal 10min, the visible transparent adhesive tape sample precipitation at the pipe end; Abandon supernatant, 75% ethanol rinsing 2 times; Air-dry, appropriate DEPC (Shanghai past bio tech ltd) water dissolution RNA.
3.2RT-PCR
Adopt synthetic cDNA the first chain of Reverse Transcription System kit reverse transcription test kit (TAKARA).Get the MDCK-HA cell total rna of 5 μ L extractings, the Auele Specific Primer that 1 μ L12bp is conservative (AGCAAAAGCAGG (SEQ ID NO:3)), 6 μ L sterilizing DEPC process water and mix, and in 70 ℃ of effect 5min, put rapidly on ice, slightly centrifugal.Add inverse transcription reaction liquid (containing 5 * reaction buffer, 4 μ L, RNase inhibitor 1 μ L, 10mM dNTP mix2 μ L), 37 ℃ of temperature are bathed 5min, then add 1 μ L M-MuLV-RNase to mix, 42 ℃ of temperature are bathed 60min, 70 ℃ of 10min deactivation 3.3 ThermoScript II.
CDNA the first chain synthesizing of take is template, as case study on implementation 1PCR amplification H5 gene.PCR product is through 1.0% agarose gel electrophoresis analysis, and as shown in Figure 4, MDCK-HA clone can amplify the specific band of 1.7Kb to result, but MDCK-HA cell total rna the uncontaminated plasmid containing H5 gene.
Embodiment 4:MDCK-HA infection of cell line H9N2 subtype avian influenza virus
Eugonic mdck cell is forwarded on 24 orifice plates, when it grows to proper concn, for virus infection, test.The H9N2 subtype avian influenza virus of the low pathogenicity of suitable infective dose and MDCK-HA cell are hatched, and three samples are repeated in every hole, connect the rear observation of cell pathology of poison, and when meeting poison rear 24h, 48h, 72h, 96h, detect the hemagglutinative titer of cells and supernatant.Meanwhile, setting up the normal mdck cell infection H9N2 of system subtype avian influenza virus is contrast.Experimental result shows, H9N2 subtype avian influenza virus can be on not containing the MDCK-HA cell in the substratum of TPCK-Trypsin well-grown, and blood clotting (Fig. 5) cannot detected on normal mdck cell.
Meanwhile, also monitor as stated above H1 (as PR8), H3 hypotype, H4 hypotype and the growing state of H10 subtype avian influenza virus in MDCK-HA clone, the results are shown in Figure 6A-6D.Result shows that these viruses all can well-grown, and cannot measure blood clotting valency on normal MDCK.
The optimization of embodiment 5 H9N2 subtype avian influenza virus proliferation conditions on rolling bottle cell
1. kind of malicious SH441/PR8 (NS2 E67 & 74S) viral level is measured
MDCK-HA cell is laid in 24 well culture plates, adds nutrient solution [DMEM, containing 5%NCS (PAA)], and be put in and in cell culture incubator, cultivate 18～22h.Treat that cell grows up to individual layer to 90%, discard nutrient solution, PBS washes three times, and discards.(extent of dilution is 10 to add viral SH441/PR8 to be measured (NS2E67 & 74S)
-3-10
-7), 100ul/ hole, each extent of dilution repeats three holes, hatches 1.5h in cell culture incubator, during every 15min, shake once, make virus fully contact with cell.Hatch after end, discard virus liquid, and PBS washes three times.Discard after PBS, add 2% calf serum DMEM nutrient solution, and be placed in cell culture incubator and cultivate.Hatch after 48h, measure the hemagglutinative titer of cell conditioned medium.According to Reed and Muench method, virus titer is calculated.
Result: plant malicious TCID
50measurement result is 10
5.75tCID
50/ 100ul.
The present invention also provides the growth curve (method is the same) of SH441/PR8 (NS2E67 & 74S) simultaneously, specifically as shown in Figure 7, the viral coagulant property of data presentation SH441/PR8 (NS2E67 & 74S) virus is better than wild malicious SH441.
2. the optimization of virus infection dosage
When MDCK-HA cell grows up to monolayer cell to 90%, discard nutrient solution, and wash 3 times with PBS.By SH441/PR8 (NS2E67 & 74S) virus strain with different infective doses (8 * 10
4, 3 * 10
4, 1.5 * 10
4, 0.8 * 10
4, 0.4 * 10
4tCID
50) be inoculated in respectively MDCK-HA cell, establish negative control simultaneously.In viruses adsorption process, every 15min, shake Tissue Culture Flask once, virus is fully contacted with cell.After viruses adsorption effect 1.5h, discard virus liquid, add 2% calf serum DMEM maintenance medium to continue to cultivate.When virus infection 24h, 36h, 48h, 60h and 72h, collect respectively cell conditioned medium and measure its hemagglutinative titer.
Result is as shown in table 1: with 0.4 * 10
4tCID
50with 0.8 * 10
4tCID
50virus infection MDCK-HA cell time, the virus titer that different time sections records is all than 1.5 * 10
4tCID
50with 3 * 10
4tCID
50a low titre; 8 * 10
4tCID
50during virus infection MDCK-HA, after infection, after 36h, record virus titer and be 2
12, and at same infection time point, than 3 * 10
4tCID
50the high titre of virus quantity of virus infected cell amplification.Consider, the virus infection dosage of selecting is 8 * 10
4tCID
50, and receive the poison time for meeting the rear 48h of poison.
Virus amplification effect under table 1 different virus infective dose
3. the maintenance medium of different varieties screening
Cultivate MDCK HA-2 cell grow to monolayer cell 90% time, discard nutrient solution, with PBS, clean 3 times.By 8 * 10
4tCID
50sH441/PR8 (NS2 E67 & 74S) inoculating cell, add respectively serum-free medium+TPCK pancreatin, serum-free medium and 2%NCS DMEM nutrient solution to hatch, separately establish negative control.When virus infection 24h, 36h, 48h, 60h and 72h, collect respectively cell conditioned medium and measure its hemagglutinative titer.Result is as shown in table 2, and during virus inoculation MDCK-HA cell 48h, the viral hemagglutinative titer that serum-free medium+TPCK pancreatin, serum-free medium and 2%NCS DMEM nutrient solution are cultivated is 2
12.After infection, during 36h, the viral hemagglutinative titer that serum-free medium (GIBCO)+TPCK pancreatin (Worthington) maintenance medium is cultivated all exceeds a virus titer than serum-free medium and 2%NCS DMEM nutrient solution.Mixed economy cost, should select 2%NCS DMEM maintenance medium to cultivate MDCK-HA cell amplification virus, and the receipts poison time is 48h.
The impact of the different viral maintenance medium of table 2 on virus amplification effect
4. the optimization of maintenance medium final volume
In 3L rolling bottle, cultivate MDCK-HA cell, until MDCK-HA grow up to monolayer cell 90% time, discard nutrient solution, with PBS, clean 3 times.By 8 * 10
4tCID
50viral dosage inoculating cell, and cultivate by the maintenance medium of 2%NCS DMEM.The final volume of maintenance medium is established two groups, is respectively 50ml and 100ml.At virus infection 6h, 12h, 24h, 36h, 48h, 60h, during 72h, measures respectively the hemagglutinative titer of respectively organizing virus in cell conditioned medium.
Result is as shown in table 3: in virus inoculation rolling bottle, after MDCK-HA cell, the virus titer that 50ml maintenance medium obviously records than 100ml maintenance medium after meeting malicious 6h is high, and 50ml maintenance medium 36h virus titer after connecing poison can reach 2
12, and 100ml maintenance medium 60h virus titer after connecing poison just reaches 2
11, therefore, rolling bottle cell connects after poison, and the viral effect of the final volume culturing cell amplification of use 50ml maintenance medium is more effective than 100ml maintenance medium.
Table 3 difference maintains the long-pending impact on virus amplification effect of liquid
In a word, after optimizing by above-mentioned parameter, on the MDCK-HA cell of SH441/PR8 (NS2 E67 & 74S) virus in rolling bottle, propagation is good, and the hemagglutinative titer of cell conditioned medium can reach 2
12, meet the requirement that vaccine candidate strain is tired.
The development of enforcement case 6 cell inactivated vaccines and immune effect assessment thereof
1. the screening of inactivator
Select formaldehyde (raw work) and two kinds of inactivators of beta-propiolactone (Wako), according to ablation method deactivation same virus separately, the inactivating efficacy of more different inactivators.The deactivation of formaldehyde: get 2 pipe 50ml centrifuge tubes, all add 20ml virus liquid, add formaldehyde eventually to concentration be 0.1%, and be put in deactivation on 37 ℃ of shaking tables.Wherein one manage virus liquid deactivation 24h, another pipe virus liquid deactivation 36h.After deactivation finishes, measure hemagglutinative titer.In addition, the virus liquid of getting deactivation is inoculated in 20 pieces of 9～11 age in days SPF chicken embryos, and 0.2mL/ piece measures inactivating efficacy.After 48h, detect the hemagglutinative titer of chick embryo allantoic liquid, as HA, test is negative, and judges that deactivation is complete, otherwise is disqualified upon inspection.The deactivation of beta-propiolactone: get the same 20ml virus liquid of 3 pipes, by 1:2000 dilution beta-propiolactone, be put in 4 ℃ of shaking table deactivations, shake respectively 4h, 8h and 12h, be then put in 37 ℃ of incubator 2h, measures hemagglutinative titer.Get in addition deactivation liquid and be inoculated in 20 pieces of 9～11 age in days SPF chicken embryos, 0.2mL/ piece, measures inactivating efficacy.After 48h, detect the hemagglutinative titer of chick embryo allantoic liquid, as hemagglutination test is negative, judge that deactivation is complete, otherwise be disqualified upon inspection.After two kinds of inactivator deactivations complete, survey respectively the hemagglutinative titer of virus liquid after deactivation, detect the impact of inactivator on virus titer.
Result demonstration, after formalin-inactivated 24h, inactivating efficacy is that deactivation is complete.After beta-propiolactone deactivation 4h, inactivating efficacy is that deactivation is complete.After beta-propiolactone deactivation, three groups of the hemagglutinative titer of virus are 2
12, be consistent with blood clotting valency before deactivation, show that HA antigen amount is substantially constant; After formalin-inactivated, the hemagglutinative titer of virus is 2
10, than low two titres of blood clotting valency before deactivation, show that HA antigen amount reduces.In sum, beta-propiolactone inactivator deactivation SH441/PR8 (NS2 E67 & 74S) for this test, inactivation time is 8h.
2. the screening of adjuvant
With common white-oil adjuvant or Montanide ISA 70VG (French SEPPIC company) respectively and SH441/PR8 (NS2 E67 & 74S) virus be prepared into deactivation vaccine, immunity SPF chicken, 3 weeks rear blood sampling separation of serum are also measured HI antibody titer.
Result shows, the HI antibody horizontal that deactivation vaccine prepared by Montanide ISA70VG adjuvant produces is prepared high 4 times than common white-oil adjuvant.Thereby the adjuvant with Montanide ISA70VG is selected in this research.
3. vaccine preparation and immune efficacy assessment thereof
By the optimum result of virus proliferation conditions on cell, the selected preferred plan higher virus of a collection of virus titer that increases, for the preparation of vaccine.First use beta-propiolactone deactivation, after testing inactivation of virus completely after, adding final concentration is 0.01% Thiomersalate (raw work), the mass ratio of virus liquid and adjuvant is 3:7.According to ordinary method, carry out emulsification: first adjuvant is put into clean beaker, 2000r/min, pre-emulsification 2min; Slowly in beaker, add a certain amount of deactivation virus liquid completely again, when adding along with virus liquid, speed of rotation also improves gradually, and after virus liquid adds completely, the speed of high-shear machine reaches 6000r/min, now starts timing 5min; During 2～3min, in tentative a moment, take out release of heat by shears, treats that temperature reduces, and emulsification is to 5min again, powered-down, quantitative separating.The check of emulsification finished product: take the clean suction pipe vaccine that takes a morsel to drip in clean tri-distilled water surface, except first, should be the indiffusion of oil droplet shape.
Result demonstration, vaccine is prepared qualified.
By laboratory development avian influenza poison cell inactivated vaccine [H9N2, SH441/PR8 (NS2E67 & 74S)] strain, to 3 week age SPF chicken (SPF chicken embryo is purchased from Beijing Cimmeria laboratory animal company, then in negative pressure isolator, raise voluntarily) carry out chest muscle immunity, 0.3mL/ is only.After immunity 3 weeks, use respectively 10
6eID
50441 viruses (laboratory preservation) and A/chicken/Shangdong/A2043/2011 (H9N2) (referred to as SDA2043 virus) (laboratory preservation) Intravenous Infection chicken.It is separated that after infecting, the 3rd day and the 5th day collection throat swab and cloaca swab carry out virus.
As shown in Figure 8, after vaccine immunity, chicken just can produce the HI antibody of anti-H9 for first week to result after immunity, and after immunity the 3rd week, the HI antibody of average anti-H9 is 2
12above.As show as shown in 4-5, avian influenza poison cell inactivated vaccine that this test is developed all can 100% ground protection chicken avoids 441 or the attack of SDA2043 virus.This result shows, this tests prepared avian influenza poison cell inactivated vaccine (H9N2, SH441HANA/PR8+NSmutant strain) can protect the chicken H9N2 subtype avian influenza virus epidemic strain of avoiding infection preferably.
Throat swab virus separating resulting after table 4 Immunization
Cloaca swab virus separating resulting after table 5 Immunization
Claims (4)
1. a spinner culture method for H9N2 subtype avian influenza virus, is characterized in that comprising the following steps:
1) step of cultivating MDCK-HA cell, is positioned over MDCK-HA cell in rolling bottle, adds nutrient solution, and rolling bottle is positioned over and in cell culture incubator, cultivates 18～22h;
2) step of inoculating H9N2 subtype avian influenza virus in MDCK-HA cell, the cell in rolling bottle grows up to monolayer cell and reaches 90% when above, discards nutrient solution, adopt PBS solution to wash 1～5 time, and discard PBS solution, and inoculation H9N2 subtype avian influenza virus, the dosage of inoculation is 8 * 104TCID50, in cell culture incubator, hatch 1～2 hour, every 10～20 minutes, shake rolling bottle once, hatch after end, discard virus liquid, adopt PBS to wash 1～5 time, then discard PBS;
3) then add 2%NCS DMEM maintenance medium to cultivate, the volume of described DMEM maintenance medium is 50ml, is placed in cell culture incubator and cultivates, and cultivates 48 hours, collects cell conditioned medium.
2. the spinner culture method of H9N2 subtype avian influenza virus as claimed in claim 1, is characterized in that, after inoculation, shakes rolling bottle every 15 minutes.
3. the spinner culture method of H9N2 subtype avian influenza virus as claimed in claim 1, is characterized in that, described H9N2 subtype avian influenza virus is A/Chicken/Shanghai/441/2009 or A/chicken/Shangdong/A2093/2012.
4. the spinner culture method of H9N2 subtype avian influenza virus as claimed in claim 3, is characterized in that: described H9N2 subtype avian influenza virus is SH441/PR8 (NS2 E67 & 74S) virus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410306875.4A CN104073470A (en) | 2014-06-30 | 2014-06-30 | Spinner-flask culture method for H9N2 subtype of avian influenza virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410306875.4A CN104073470A (en) | 2014-06-30 | 2014-06-30 | Spinner-flask culture method for H9N2 subtype of avian influenza virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104073470A true CN104073470A (en) | 2014-10-01 |
Family
ID=51595077
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410306875.4A Pending CN104073470A (en) | 2014-06-30 | 2014-06-30 | Spinner-flask culture method for H9N2 subtype of avian influenza virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104073470A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105816870A (en) * | 2016-03-24 | 2016-08-03 | 福州大北农生物技术有限公司 | Preparation method of avian influenza virus inactivating vaccine |
| CN108220221A (en) * | 2017-12-22 | 2018-06-29 | 吉林冠界生物技术有限公司 | A kind of method of full suspension cell culture recombinant fowl influenza subtype virus |
| CN111454915A (en) * | 2020-03-10 | 2020-07-28 | 成都天邦生物制品有限公司 | Separation and identification method for H9 subtype avian influenza virus |
| CN112294952A (en) * | 2019-08-02 | 2021-02-02 | 成都天邦生物制品有限公司 | Triple inactivated vaccine for newcastle disease, H9 subtype avian influenza and avian adenovirus |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914498A (en) * | 2010-02-08 | 2010-12-15 | 中国农业科学院上海兽医研究所 | MDCK (Madin-Darby Canine Kidney) cell line for stably expressing highly pathogenic avian influenza virus (HPAIV) HA (Hemagglutinin) protein and application thereof |
| CN101979517A (en) * | 2010-10-15 | 2011-02-23 | 洛阳普莱柯生物工程有限公司 | Method for producing influenza viruses in large scale by using bioreactor |
| CN102657859A (en) * | 2012-05-17 | 2012-09-12 | 肇庆大华农生物药品有限公司 | Method for producing avian influenza vaccine by using cells cultured by spherical microcarrier bioreactor |
| CN102676460A (en) * | 2012-06-19 | 2012-09-19 | 肇庆大华农生物药品有限公司 | Method for vaccinating avian influenza virus through microcarrier suspension culture cell |
-
2014
- 2014-06-30 CN CN201410306875.4A patent/CN104073470A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914498A (en) * | 2010-02-08 | 2010-12-15 | 中国农业科学院上海兽医研究所 | MDCK (Madin-Darby Canine Kidney) cell line for stably expressing highly pathogenic avian influenza virus (HPAIV) HA (Hemagglutinin) protein and application thereof |
| CN101979517A (en) * | 2010-10-15 | 2011-02-23 | 洛阳普莱柯生物工程有限公司 | Method for producing influenza viruses in large scale by using bioreactor |
| CN102657859A (en) * | 2012-05-17 | 2012-09-12 | 肇庆大华农生物药品有限公司 | Method for producing avian influenza vaccine by using cells cultured by spherical microcarrier bioreactor |
| CN102676460A (en) * | 2012-06-19 | 2012-09-19 | 肇庆大华农生物药品有限公司 | Method for vaccinating avian influenza virus through microcarrier suspension culture cell |
Non-Patent Citations (6)
| Title |
|---|
| 任超超等: "利用反向遗传技术构建细胞高适应性的H9N2流感病毒", 《中国动物传染病学报》 * |
| 徐大伟: "H9N2亚型禽流感病毒与鸭坦布苏病毒的生物学特性研究及疫苗的初步研制", 《万方数据库》 * |
| 谢秋玲等: "昆虫细胞Sf9在四种生物反应器中的培养", 《暨南大学学报（自然科学版）》 * |
| 赵佼等: "转瓶内部结构对无血清悬浮培养昆虫细胞的影响", 《生物工程学报》 * |
| 陈圆等: "H9N2亚型禽流感病毒灭活苗最小免疫剂量及抗体消长规律的初步研究", 《中国动物传染病学报》 * |
| 雷雯等: "细胞转瓶培养技术研究进展", 《中国兽药杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105816870A (en) * | 2016-03-24 | 2016-08-03 | 福州大北农生物技术有限公司 | Preparation method of avian influenza virus inactivating vaccine |
| CN108220221A (en) * | 2017-12-22 | 2018-06-29 | 吉林冠界生物技术有限公司 | A kind of method of full suspension cell culture recombinant fowl influenza subtype virus |
| CN112294952A (en) * | 2019-08-02 | 2021-02-02 | 成都天邦生物制品有限公司 | Triple inactivated vaccine for newcastle disease, H9 subtype avian influenza and avian adenovirus |
| CN111454915A (en) * | 2020-03-10 | 2020-07-28 | 成都天邦生物制品有限公司 | Separation and identification method for H9 subtype avian influenza virus |
| CN111454915B (en) * | 2020-03-10 | 2023-11-10 | 成都史纪生物制药有限公司 | H9 subtype avian influenza virus separation and identification method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Continued evolution of H5N1 influenza viruses in wild birds, domestic poultry, and humans in China from 2004 to 2009 | |
| Xu et al. | Phylogenetic classification of hemagglutinin gene of H9N2 avian influenza viruses isolated in China during 2012–2016 and evaluation of selected candidate vaccine strains | |
| Heinen | Swine influenza: a zoonosis | |
| Chen et al. | Molecular and antigenic characteristics of Massachusetts genotype infectious bronchitis coronavirus in China | |
| CN104232594B (en) | Recombination classes fowl type H1N1 inactivated influenza virus vaccines strain (JS40/PR8) and its preparation method and application | |
| Choi et al. | Genetic relationship of H3 subtype avian influenza viruses isolated from domestic ducks and wild birds in Korea and their pathogenic potential in chickens and ducks | |
| Xu et al. | Isolation and characterization of an H9N2 influenza virus isolated in Argentina | |
| CN102220293B (en) | Dog flu recombinant virus as well as preparation method and application thereof | |
| CN104073470A (en) | Spinner-flask culture method for H9N2 subtype of avian influenza virus | |
| CN103328629A (en) | Novel vaccines against the a/H1N1 pandemic flu virus | |
| CN104338127A (en) | Method for producing inactivated vaccine of H9N2 subtype of avian influenza virus and product of inactivated vaccine | |
| CN103275939B (en) | Recombinant virus of chimeric IBV H120 S1 gene ectodomain suitable for cell culture and construction method and application thereof | |
| CN103937753B (en) | H9N2 subtype avian influenza virus strain and inactivated vaccine thereof and application | |
| CN103614345B (en) | A kind of influenza virus vaccine strain | |
| Xu et al. | Epidemiology, evolution, and biological characteristics of H6 avian influenza viruses in China | |
| CN104830811A (en) | NS1 gene deleted and live-attenuated vaccine candidate strain of H9N2 subtype avian influenza virus and its establishing method and application | |
| CN104164408B (en) | Anti-newcastle disease, infectious bronchitis and avian influenza vaccine compositions and preparation | |
| CN108300702B (en) | Chicken-derived H9N2 avian influenza virus cold-adapted strain screening method and application thereof | |
| CN107353328A (en) | A kind of H9N2 subtype avian influenza virus sample particles of restructuring and its production and use | |
| CN109266623A (en) | One strain vaccine strain rSHA- △ 200 and its construction method and application | |
| CN100467591C (en) | Recombinant fowl influenza virus strain, its preparation method and the vaccine obtained therefrom | |
| CN105087502B (en) | H9N2 subtype avian influenza virus temperature sensitivity attenuated live vaccine Candidate Strains CA 28（AH）And its application | |
| RU2507256C2 (en) | Strain of influenza virus a/17/mallard/netherlands/00/95(h7n3) for production of live and inactivated influenza vaccines | |
| CN102234637B (en) | Preparation for reconstruction influenza A H1N1 virus inactivated vaccine strain (SC/PR8), and use thereof | |
| CN101914500A (en) | Gene-recombined H9N1 subtype avian influenza virus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20141001 |