CN110527659A - For producing the suspension cell strain of influenza vaccines - Google Patents
For producing the suspension cell strain of influenza vaccines Download PDFInfo
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Abstract
The present invention relates to a kind of for producing the suspension cell strain of influenza vaccines, and the cell strain is MDCK-G1XF cell strain, is preserved in China typical culture collection center, and deposit number is CCTCC NO:C2019112.The invention further relates to the application of the cultural method of the cell strain and the cell in culture influenza virus or preparation influenza vaccines, it is preferred that the influenza virus is H5N1 virus.
Description
Technical field
The invention belongs to field of biotechnology, in particular to a kind of for producing the suspension cell strain of influenza vaccines.
Background technique
Currently, widely used influenza vaccines are trivalent and tetravalence influenza combined vaccine both at home and abroad, pair of prevention effect is played
As comprising two kinds of A types and one or two kinds of influenza B virus, the load that chicken embryo is inoculated with by these vaccines as influenza virus mostly
Body is produced[1].But its presence is also found by production all the year round really using chicken embryo as the main carriers of production influenza vaccines
Many drawbacks: the limitation of its raw material, the residual of impurity protein, more verbose process route etc., these drawbacks all limit
The development of influenza vaccines.These offshore companies have formd the complete production flowing water produced using mdck cell as matrix
Line, and listed[2,3].In contrast, though domestic have more document to expand further investigation to this aspect, fail shape
At more mature cellular matrix influenza vaccines production line, and the serum free suspension domestication of the mdck cell of the first step is outstanding among these
For key, the property of cell strain directly determines the quality and yield of vaccine.Root it is documented that, there are a small number of public affairs in the present country
There has been relatively stable suspension cell strain in department, and there has also been obvious for domestic mdck cell serum free suspension cell technology in recent years
Development[4]。
Domestic influenza vaccines are all still produced using traditional chicken embryo technique now, but produce influenza epidemic disease using chicken embryo
Seedling is implicitly present in many shortcomings, for example while carrying out preparation influenza vaccines using chicken embryo technique is difficult to realize be fully automated,
It is artificial plus semi-automatic assembly line mostly；The excessive cycle of chicken embryo is cultivated, the production cycle of general a batch vaccine is two weeks
Left and right；In each process especially in the step great work intensity for isolating and purifying effective component from allantoic fluid；Simultaneously
It is not easy to guarantee the quality stability of different batches[5,6].In bird flu high-incidence period, but will because of chicken class a large amount of infection and lack
The weary raw material for preparing vaccine.In consideration of it, with mammalian cell replace chicken embryo as production production influenza vaccines carrier,
To prepare influenza vaccines have become we there is an urgent need to because of chicken embryo when can not only avoid bird flu great outburst completely in this way
Shortfall risk, while can also remove the impurity components such as the chicken embryo ovalbumin in vaccine, then reduce caused by vaccine inoculation
Some allergic reactions[7]。
In recent years, external certain listed a company some influenza vaccines for using zooblast as matrix, referred to as cell
Matrix influenza vaccines, MDCK cell (Madin-Darby canine kidney cells) is that the kidney derived passage of normal dogs is thin
Born of the same parents.Madin and Darby in 1958 is separately cultured foundation from the renal tissue of healthy adult bitch (Cocker Spaniel),
Continuous cell line is formed by conversion[8].Mdck cell majority currently used for research derives from ATCC, due to mdck cell
It is known as being to be most suitable for the cellular matrix of influenza vaccines production, therefore be used to produce influenza epidemic disease by lot of domestic and international producer
Seedling, and achieve preferable achievement[9], and it is used to produce vaccine and mainly passes through sheet or microcarrier or complete serum-free
Single-cell suspension mode carries out the culture of mdck cell, and above-mentioned several relatively conventional mdck cell training methods are used to carry out
Again more advantageous with serum-free single-cell suspension culture in the production of cellular matrix influenza vaccines, reason is production cost
It reduces and the increasing of unit volume cell concentration, virus infection area increases, to make yield increase, produce effect and increase[10,11]。
Applicant is first to work it has been reported that the strain of MDCK-G1 sub-types of cells is as suitable for the production of H5N1 influenza
MDCK subcellular strain (CN201710676196.X), by safety research, G1 cell strain carries out internal cellular neoplastic, thin
The carcinogenicity of cellular lysate object and DNA detection etc. shows that MDCK-G1 subcellular strain has low oncogenicity, quick to H5N1 influenza height
Sense has the Potential feasibility for Pandemic influenza vaccine production.But there is still a need for addition serum for the culture of the cell strain
With cell attachment carrier, it is unfavorable for industrialized production of vaccine, therefore, it is necessary to further be tamed to it, obtains more
Suitable for industrializing the serum free suspension cell strain of amplification.
Bibliography:
[1]Castro R,Fernandes P,Laske T,et al.Production of canine adenovirus
type 2 in serum-free suspension cultures of MDCK cells[J].Appl Microbiol
Biotechnol,2015,99(17):7059-7068.
[2]Shin D,Park K J,Lee H,et al.Comparison of immunogenicity of cell-
and egg-passaged viruses for manufacturing MDCK cell culture-based influenza
vaccines[J].Virus Res,2015,204:40-46.
[3]Looi Q H,Foo J B,Lim M T,et al.How far have we reached in
Development of effective influenza vaccine [J] .Int Rev Immunol, 2018,37 (5):
266-276.
[4]Capra J,Eskelinen S.Correlation between E-cadherin interactions,
survivin expression,and apoptosis in MDCK and ts-Src MDCK cell culture models
[J].Lab Invest,2017,97(12):1453-1470.
[5]Cooper K,Viswanathan C.Establishment of a mesenchymal stem cell
bank[J].Stem Cells Int, 2011,2011:905621.
[6]Gu C,Li P,Liu W,et al.The role of insulin in transdifferentiated
hepatocyte proliferation and function in serum-free medium[J].J Cell Mol Med,
2019.
[7]Huang D,Peng W J,Ye Q,et al.Serum-Free Suspension Culture of MDCK
Cells for Production of Influenza H1N1 Vaccines[J].PLoS One,2015,10(11):
e141686.
[8]Madin SH,Darby NB,Jr.:Established kidney cell lines of normal
adult bovine and ovine origin.Proc Soc Exp Biol Med 1958,98(3):574-576.
[9]Romanovskaia A A,Durymanov A M,Sharshov K A,et al.[Investigation
of susceptibility of influenza viruses A(H1N1),the cause of infection in
humans in April-May 2009,to antivirals in MDCK cell culture][J]. Antibiot
Khimioter,2009,54(5-6):41-47.
[10]Vignola R,Misto R,Giurgola L,et al.A two-centre validation study
of sterility test of corneal storage media with elimination of interfering
antimicrobials in compliance with the European Pharmacopoeia[J].Cell Tissue
Bank,2019.
[11]Jordan I,Lohr V,Genzel Y,et al.Elements in the Development of a
Production Process for Modified Vaccinia Virus Ankara[J].Microorganisms,2013,
1(1):100-121.
Summary of the invention
Present invention firstly relates to one plant of MDCK subcloned cells strain MDCK-G1XF cell strain, the cell strain is preserved in
China typical culture collection center, deposit number are CCTCC NO:C2019112, address, Wuhan City, Hubei Province Wuchang District eight
All the way No. 299 Wuhan Universitys in the school, postcode 430072, phone 027-68752319.
The invention further relates to the cultural methods of the MDCK-G1XF cell strain, and the method includes following parameters:
(1) culture medium of the cell is serum free medium；
(2) by the cell inoculation in culture medium, in the culture that suspends in culture vessel；
(3) the multiplication period of the cell is 48 hours.
Preferably, the culture medium be serum free medium (each component content is as follows in serum free medium specific formula:
D-Glucose 4500-9000mg/l, Sodium Pyruvate 60-200mg/l, l-Alanine 10-45mg/l, L-arginine 150-300mg/
L, altheine 20-45mg/l, L-Aspartic acid 30-65mg/l, l-cysteine 30-45mg/l, L-cysteine 20-
80mg/l, Pidolidone 30-60mg/l, L-Glutamine 350-1000mg/l, glycine 30-80mg/l, L-Histidine 50-
120mg/l, l-Isoleucine 80-120mg/l, L-Leu 100-160mg/l, L-lysine 100-200mg/l, L- first sulphur
Propylhomoserin 50-100mg/l, L-phenylalanine 100-140mg/l, L-PROLINE 50-100mg/l, Serine 50-100mg/l,
L-threonine 120-150mg/l, L-Trp 50-100mg/l, l-tyrosine 50-80mg/l, Valine 100-150mg/l,
Hypoxanthine 1-15mg/l, thymidine 0.2-1mg/l, D-Biotin 0.005-0.1mg/l, folic acid 2-5mg/l, niacinamide 2-4mg/
L, pyridoxol 2-4mg/l, thiamine 1-5mg/l, riboflavin 0.1-0.5mg/l, choline chloride 10-30mg/l, D-VB5 calcium 2-
5mg/l, inositol 10-30mg/l, sodium selenite 40-80mg/l, ferric nitrate 10-30mg/l, ferrous sulfate 0.1-0.5mg/l, sulphur
Sour magnesium 20-50mg/l, potassium chloride 250-400mg/l, sodium chloride 5000-8000mg/l, disodium hydrogen phosphate 40-80mg/l, phosphoric acid
Sodium dihydrogen 40-80mg/l, sodium bicarbonate 2000-3000mg/l, magnesium chloride 1000-3000mg/l, calcium chloride 100-200mg/l,
Zinc sulfate 0.2-1mg/l, copper sulphate 10-20mg/l, insulin 2-10mg/l, soy hydrolyzate 2000-3000mg/l, ethanol amine
2-5mg/l, glutathione 1-5mg/l, ferric citrate 15-30mg/l, blocked polyethers F68 200-2000mg/l, Anti-
Clumping agent 5ml/l, cholesterol 3-5mg/l, phenol red 5-10mg/l), pH 7.0-7.4；
The culture vessel is the Multiple Types shaking flasks such as 125ml, 250ml, 500ml, 1L or industrial culture device；
The culture vessel is placed in shaking table culture, it is 130rpm that shaking table, which shakes speed,；
Condition of culture is 37 DEG C, and CO2 concentration is 5%；
1 × 10^6/ml of initial inoculation concentration of the cell.
The invention further relates to application of the MDCK-G1XF cell strain in the product of preparation prevention or treatment influenza.
The invention further relates to using the MDCK-G1XF cell strain as carrier, the method for cultivating influenza virus, feature exists
In described method includes following steps:
It (1) is 3.0~5.0 × 10 in the cell density6When cells/ml, it is inoculated with and is flowed with MOI 0.00001~0.001
Influenza Virus；
(2) replacement cell culture fluid is virus-culturing fluid after being inoculated with, and culture to HA titre plateau harvests virus liquid；
(3) centrifugation removal cell impurities are carried out to virus liquid and harvests influenza virus.
Preferably,
The influenza virus is H5N1 virus；
Most suitable kind poison amount is 0.5ml/40ml when inoculation, while adding the TPCK pancreatin that concentration is 8 μ g/ml；
Cell density described in step (1) is 4 × 106Cells/ml, the MOI are 0.0001.
The beneficial effects of the present invention are: the method that the present invention uses serum free suspension domestication, successfully having obtained can be in nothing
The cell strain that serum suspended state is well grown, and this MDCK-G1XF cell strain is able to maintain even enhancing infected by influenza
(H5N1) sensibility.Mdck cell at present foreign countries get the Green Light can be used for influenza vaccines production, but China still
In the research and development primary stage, therefore without national examination criteria, we refer to the existing pharmacopoeial requirements in China, with reference to the world
On latest requirement, to we tame MDCK-G1XF cell carried out the sensitivity testing that influenza virus is infected.Detection project
Mainly comparison two kinds of cell strains of MDCK-G1 and MDCK-G1XF are to H5N1 influenza sensibility to the preliminary assessment MDCK
The practicability that suspension cell strain is produced as H5N1 influenza vaccines.
Due to the MDCK-G1XF cell strain for newly filtering out, experiments verify that, it is most suitable when by the inoculation of MDCK-G1XF
Kind poison amount be 0.5ml/40ml, TPCK pancreas enzyme concentration be 8 μ g/ml, the virus liquid titre at this moment harvested at 73h (3d) most
High reachable 1:256.And in contrast, it is 1:64- that H5N1 is inoculated in its titre of virus liquid harvested after adherent MDCK-G1 cell
1:128.To sum up, the MDCK-G1XF cell strain that we filter out have it is highly sensitive to H5N1 influenza, have for being very popular
The potentiality of influenza vaccines production.
Detailed description of the invention
The continuous 50 generation upgrowth situation figure of MDCK-G1 plants of cells (Figure 1A-Fig. 1 E) when Fig. 1, serum free suspension domestication.
According to the different generation mirror following figures of domestication sequence in the suspension domestication of Fig. 2, MDCK-G1 cell.
Fig. 3, MDCK-G1XF cell detection of mycoplasma DNA decoration method result figure, Fig. 3 A: positive control, Fig. 3 B:MDCK-
G1XF cell.
Titre situation of change (the legend generation of virus liquid is harvested after Fig. 4, MDCK-G1XF cell inoculation H5N1 under different condition
The different pancreas enzyme concentration of table, unit are μ g/ml), Fig. 4 A: kind poison amount 1ml/40ml, Fig. 4 B: kind poison amount 0.5ml/40ml, Fig. 4 C:
Kind poison amount 0.25ml/ml, 4D: kind poison measures 0.125ml/40ml).
The titer determination of virus liquid is harvested after Fig. 5, MDCK-G1 cell inoculation H5N1.
Specific embodiment
Main material:
Cell: MDCK-G1XF cell, deposit number are CCTCC NO:C2019112, preservation date, May 14 in 2019
Day, specific name are as follows: dog kidney suspension cell MDCK-G1XF.Address, No. 299 Wuhan of Wuhan City, Hubei Province Wuchang District Bayi Road are big
In school, postcode 430072, phone 027-68752319.It is the H5N1 influenza height that applicant passes through that the screening that formerly works obtains
The MDCK subcellular strain of sensitive, the low tumorigenesis of degree.
Virus: H5N1 avian influenza virus vaccine strain: strain name NIBRG-14 is introduced from NIBSC, the fowl that WHO recommends
Influenza strain.Chicken embryo H5N1 totivirus inactivated influenza vaccine is prepared by this Laboratory Production.
Main agents: DMEM culture medium is purchased from Gibco company, article No.: 12100061；Fetal calf serum is purchased from Gibco company,
Article No.: 16140071；Pancreas junket soya peptone fluid nutrient medium, THIOGLYCOLLIC ACID salt broth are trained by Wuhan Biological Products Inst.
Support basal cell's preparation.TPCK-trypsin is purchased from Sigma company.
Mdck cell library is established: one MDCK-G1 cell strain of recovery, conventional to establish preceding master library, master library and work library.
H5N1 influenza work Virus seed library is established: the strain NIBRG-14 for taking a NIBSC to introduce is inoculated in 11 age in days SPF
Chicken embryo, the chicken embryo after inoculation are placed in 33 DEG C of culture 72h, then chicken embryo Cool Room 4 DEG C freeze overnight and harvest allantoic fluid.Harvest is urinated
It is work Virus seed library that cyst fluid, which is distributed into 1ml/ branch,.
Embodiment 1, MDCK-G1 adhered state gradually reduce serum primary dcreening operation
The MDCK-G1 cell for taking adherent growth state more stable carries out preliminary adherent drop serum domestication, first will be normal
The culture medium of the MDCK-G1 cell of growth is changed to serum free medium, and the FBS that when beginning is still first added 10% guarantees nutritional sufficiency
Supply, observes cell growth condition every 12h, main detection its growth convergence degree, cellular morphology and growth rhythm.
If cell, which is grown under the conditions of 1:4 passage in 72h it, which grows convergence degree, can rise to 80% or more, and form it is normal,
Extensibility preferably, motility rate it is higher when, can be according to following drop serum scheme: fetal calf serum successively decrease 10% by concentration gradient
→ 8% → 6% → 5% → 4% → 3% → 2% → 1.5% → 1% → 0.8% → 0.5% → 0, the premise successively decreased is cell
Continuous 3 generation growth is stablized.If there is unstable situation (Cell viability drop in cell growth state when serum is down to a certain concentration
It is low), then it needs to carry out passage again in this concentration to adapt to it gradually, it is dense that reduction serum can be continued after its growth adaptation
Degree.
The suspension domestication and screening of embodiment 2, MDCK-G1 cell
It, will after the well-grown MDCK-G1 cell of free serum culture is digested according to conventional cell passage step
It is added in serum free medium after being centrifuged, and is inoculated in and starts to carry out suspension domestication on shaking table in the small shaking flask of 125ml.Cha Wen
Culture volume accounting is more appropriate to a quarter 1/5th in shaking flask known to offering.Suspension cell inoculum density is if spy
Very refer to being 1 × 10^6/ml.Shaking table parameter regulation is 37 DEG C of temperature, CO2 concentration 5%, revolving speed 130rpm.
It according to initial cell density is 10^6/ml when cell passes on, sampling in every 24 hours, which counts, simultaneously observes its growth shape
Condition needs to pay special attention to the Parameters variations such as its motility rate, conglomeration rate, cell dia, and is in time passed on, if cell state is good
Passage good then that different modes are carried out according to cell density, at the same guarantee culture medium nutritional sufficiency and remove it is therein it is bad because
Son makes it be in a comparison and adapts in the environment of its growth.
Screening conditions: in cell growth process, if observing, there is apparent motility rate and reduces (specific manifestation in cell growth
To there is a large amount of cell fragment), there is variation (especially form be not or not conglomeration rate significantly raised (> 30%) or cellular morphology
Rule) or the density of cell when cannot reach density multiplication within 48 hours, need to carry out in these cells screening discard or
It gives subsequent specially treated mode to correct, the screening through more generations, which is found, can more adapt to the thin of serum free suspension environment
Born of the same parents.
Passage is tamed in the suspension of embodiment 3, mdck cell
There is the processing mode of required progress when various situations during mdck cell suspension growth, usually in cell
Different passage modes is taken to be inoculated in it in suitable culture environment in a suitable density when passage.It is specific as follows:
When its 48h inner cell density cannot 3 times of growths when, if also unstable rule can discard its cellular morphology simultaneously, if cellular morphology is still
Can, then can take centrifugation be resuspended passage mode, guarantee its nutritional sufficiency supply, tame early period it is immature when can carry out more
Centrifugally operated.When it is in 48h, cell density cannot even double, if Cell viability is lower, (visible more cell is broken under mirror
Piece) can then take stand at low temperature settle method cell fragment and cell are separated.When discovery cell dispersivity is poor under its mirror
(conglomeration rate is excessively high or even more than half or forms the agglomerate of 5 cells or more) is considered as going biggish cell mass when passing at this time
It removes, usable cell strainer is filtered operation, since mdck cell diameter is about 12-15 microns, is considered as 40
The strainer of micron is filtered the larger cell mass of 4 cells of removal or more composition.Centrifugation weight can also be taken in other situations
Outstanding a variety of method passages such as method and standing sedimentation method are come the stabilization and adaptation of growing environment when guaranteeing its passage.
Embodiment 4, sterility test and detection of mycoplasma test
Sterility test: THIOGLYCOLLIC ACID salt fluid culture within validity period, having verified that qualification is got from Culture Medium Laboratory
(wherein THIOGLYCOLLIC ACID salt broth is mainly used for for base and pancreas junket soya peptone fluid nutrient medium both sterility test cultures
The culture of anaerobic bacteria, it can also be used to aerobic bacteria culture；Pancreas junket soya peptone fluid nutrient medium is used for the culture of fungi and aerobic bacteria),
Aseptic experiment is carried out, two cell samples are respectively taken from MDCK-G1 cell strain and MDCK-G1XF cell strain, according to " middle traditional Chinese medicines
Allusion quotation " Sterility Test in 2015 editions tests, it the results are shown in Table 1.
1 cell sterility test result of table
Detection of mycoplasma test: mycoplasma broth cultivation within validity period, having verified that qualification is got from Culture Medium Laboratory
Base and mycoplasma arginini broth bouillon are supported, detection of mycoplasma test (every culture medium inoculated cell suspension sample 0.5 is carried out
~1.0ml sets and cultivates 3 weeks under 37 DEG C of environment, respectively taken from 4 mycoplasma fluid nutrient mediums behind the 1st week after inoculation 2 into
Row second generation culture, every culture medium distinguish transferred species to corresponding mycoplasma semifluid culture medium and mycoplasma fluid nutrient medium each 2
Branch, sets 37 DEG C of culture days, observes 1 time every 3 days), while carrying out while carrying out cultivation and indicator cells cultivation (DNA dye
Color method), two cell samples are respectively taken from MDCK-G1 cell strain and MDCK-G1XF cell strain, according to " Chinese Pharmacopoeia " 2015 editions
Cultivation and DNA decoration method are tested, and the results are shown in Table 2.
2 detection of mycoplasma result (cultivation) of table
The determination of embodiment 5, MDCK-G1XF cell inoculation H5N1 condition
By the MDCK-G1XF cell massive amplification in shaking flask into 40 small shaking flasks of 125ml, the cell in each shaking flask is outstanding
Liquid total amount be 40ml, when the cell density in shaking flask reaches 4 × 10^6/ml, thereto be added inequality seed culture of viruses and
Then TPCK pancreatin is placed on the culture that suspends in 33 DEG C of environment.12h after virus inoculation, for 24 hours, 36h, 48h,
1ml cell suspension is taken when 60h, 72h, 84h, 96h from each shaking flask, after 1000rpm, 5min centrifugation, takes supernatant.It will be above-mentioned
Acquired supernatant carries out hemagglutination test to detect virus quantity and its invasive ability after inoculation in the cell suspension of different time.
Experimental result is shown in Fig. 4 A- Fig. 4 D, from experimental result it can be gathered that kind poison amount H5N1 most suitable when being inoculated on MDCK-G1XF cell
For 0.5ml/40ml, TPCK pancreas enzyme concentration is 8 μ g/ml, and the virus liquid titre at this moment harvested reaches as high as 1 at 73h (3d):
256。
Embodiment 6, MDCK-G1XF cell and MDCK-G1 cell compare H5N1 sensibility
After taking MDCK-G1 plants of cell adhere-wall cultures it is equally inoculated with H5N1, and harvests and be centrifuged after 72 hours and will obtain
Virus liquid carries out Hemagglutination titer measurement, by the MDCK-G1XF kind poison blood clotting Comparative result in experimental result and upper example.MDCK-G1
Harvest liquid titer determination result such as Fig. 5 after cell kind poison.
It is 1:64-1:128 that inoculation H5N1 measures its virus harvest liquid titre after adherent MDCK-G1 cell.
Titre results in the experiment of the MDCK-G1XF cell kind poison condition of front portion are compared, are supported in suspension cell
H5N1 titre is significantly higher than attached cell.Therefore the influenza virus of MDCK-G1XF cell is sensitive after serum free suspension is tamed
Property is not adversely affected, and is increased to a certain extent instead.
Finally, it should be noted that above embodiments only help skilled in the art to understand essence of the invention, do not make
For limiting the scope of the present invention.
Claims (8)
1. one plant of MDCK subcloned cells strain MDCK-G1XF cell strain, the cell strain is preserved in Chinese Typical Representative culture guarantor
Hiding center, deposit number are CCTCC NO:C2019112, address, No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road
In the school, postcode 430072, phone 027-68752319.
2. the cultural method of MDCK-G1XF cell strain according to claim 1, the method includes following parameters:
(1) culture medium of the cell is serum free medium；
(2) by the cell inoculation in culture medium, in the culture that suspends in culture vessel；
(3) the multiplication period of the cell is 48 hours.
3. according to the method described in claim 2, it is characterized in that,
The culture medium is serum free medium, pH 7.0-7.4；
The culture vessel is the Multiple Types shaking flasks such as 125ml, 250ml, 500ml, 1L or industrial culture device；
The culture vessel is placed in shaking table culture, it is 130rpm that shaking table, which shakes speed,；
Condition of culture is 37 DEG C, and CO2 concentration is 5%；
1 × 10^6/ml of initial inoculation concentration of the cell.
4. application of the MDCK-G1XF cell strain described in claim 1 in the product of preparation prevention or treatment influenza.
5. using MDCK-G1XF cell strain described in claim 1 as carrier, the method for cultivating influenza virus, which is characterized in that institute
The method of stating includes the following steps:
It (1) is 3.0~5.0 × 10 in the cell density6When cells/ml, influenza disease is inoculated with MOI0.00001~0.001
Poison；
(2) replacement cell culture fluid is virus-culturing fluid after being inoculated with, and culture to HA titre plateau harvests virus liquid；
(3) centrifugation removal cell impurities are carried out to virus liquid and harvests influenza virus.
6. according to the method described in claim 5, it is characterized in that, the influenza virus is H5N1 virus.
7. according to the method described in claim 5, it is characterized in that, most suitable kind poison amount is 0.5ml/40ml when virus inoculation, together
Shi Tianjia concentration is the TPCK pancreatin of 8 μ g/ml.
8. according to any method of claim 5-7, which is characterized in that cell density described in step (1) is 4 ×
106Cells/ml, the MOI are 0.0001.
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