CN111662882A - Method for proliferating avian influenza virus by MDCK cell line - Google Patents
Method for proliferating avian influenza virus by MDCK cell line Download PDFInfo
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- CN111662882A CN111662882A CN202010617075.XA CN202010617075A CN111662882A CN 111662882 A CN111662882 A CN 111662882A CN 202010617075 A CN202010617075 A CN 202010617075A CN 111662882 A CN111662882 A CN 111662882A
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Abstract
The invention relates to a method for proliferating avian influenza virus by adopting an MDCK cell line, which comprises the steps of acclimatizing adherent MDCK cells into a stably proliferated serum-free full-suspension culture type MDCK cell line, then inoculating the MDCK cell line into a bioreactor to complete primary cell culture, preparing avian influenza H5N1 subtype virus seed virus for suspension MDCK cell culture, inoculating the virus seed virus into suspension MDCK cells to culture, and separating and purifying virus particles. The cells obtained by domestication of the invention have stable passage, good cell morphology and high quality, can maintain the sensitivity and biological characteristics to viruses, have strong virus multiplication capacity, and can effectively multiply avian influenza virus strains.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for proliferating avian influenza virus by adopting an MDCK cell line.
Background
Avian Influenza (Bird Flu or Avian Influenza) is an acute infectious disease caused by a subtype of Influenza a virus (also called Avian Influenza virus), seriously threatens the breeding industry and human health, and brings great loss to national economy. To date, no ideal therapeutic drug for avian influenza has been found, and vaccination remains the most effective means for preventing the occurrence and prevalence of epidemic diseases today.
The traditional chick embryo process for preparing the avian influenza vaccine has a plurality of problems, such as long production period, complex operation, large workload, easy pollution, and great problems in chick embryo supply in case of large-scale outbreak of avian influenza. The cell is taken as a medium to be the current trend of vaccine preparation, and canine kidney (Madin-Darby canine kidney cells, MDCK) cells are established by Madin and Darby separation culture, are easy to culture, are rapid to proliferate, have high influenza virus infection efficiency, and are recognized cell lines which are most suitable for avian influenza virus culture at home and abroad. They are usually grown in adherent culture, with cells abutting or mosaiced, and assembling to form tight junctions on the surface, forming a monolayer of adherent cells. However, this culture method has some drawbacks, such as the adherent culture is limited by the surface area of the substrate resulting in a decrease in virus production; the procedures of liquid changing and the like in the adherent culture process are complex and tedious, the use of serum is easily polluted by microorganisms, viruses, mycoplasma and the like, and the serum-free culture technology is an effective tool for clarifying basic problems of cell generation, proliferation, differentiation and gene expression regulation, but the serum-free adherent culture is only suitable for experimental research, is not suitable for large-scale culture and cannot meet the actual production requirements of virus vaccines and the like.
Therefore, it is imperative to find a method for propagating avian influenza virus instead of the conventional process, and it is necessary to provide a method for virus amplification with good effects on cell growth rate, cell density and cell morphology, and simultaneously optimize the propagation conditions of virus in cells, so that the virus can be produced in a large scale.
Disclosure of Invention
Aiming at the defects in the prior art, the invention domesticates MDCK cells to adapt to full suspension condition culture by a method of gradually reducing serum to obtain serum-free full suspension culture type MDCK cells which can be continuously subcultured.
In order to realize the purpose of the invention, the following technical scheme is adopted: a method for propagating avian influenza virus by using MDCK cell line comprises the following steps:
step one, mixing DMEM, F12 and RPMI1640 uniformly according to a certain proportion, taking the mixture as a basic culture medium, adding FBS, recovering and culturing adherent MDCK cells, and digesting and passaging after the cells grow to a compact monolayer; gradually reducing the FBS proportion to 0%;
step two, performing shake bed expansion culture on the well-grown serum-free cultured MDCK cells until the well-grown serum-free full-suspension culture MDCK cells become acclimatized into a stably-proliferated serum-free full-suspension culture MDCK cell line;
step three, inoculating the domesticated MDCK cell line into a bioreactor to complete primary cell culture;
inoculating the avian influenza H5N1 subtype virus solution propagated by the chick embryo to a compact single-layer adherent MDCK cell to obtain a seed virus for culturing the suspension MDCK cell;
step five, inoculating the avian influenza virus seed virus to a suspension MDCK cell for culture;
and step six, separating and purifying virus particles.
Preferably, the DMEM, the F12 and the RPMI1640 are uniformly mixed according to the proportion of 2-3: 1-2: 0.5-1 to be used as a basic culture medium.
Further, DMEM, F12 and RPMI1640 are uniformly mixed according to the ratio of 3: 1 to serve as a basic culture medium.
Preferably, the digestion passage is performed using 0.25% EDTA-pancreatin.
Preferably, the H5N1 subtype virus solution is cultured in a virus maintenance solution when inoculated into adherent MDCK cells.
Further, the virus maintenance solution comprises the following components in mg/L: serine 250, arginine 250, leucine 55, isoleucine 250, alanine 2, tyrosine 55, asparagine 25, aspartic acid 25, cysteine hydrochloride 55, glutamic acid 50, glutamine 50, histidine 250, lysine 250, phenylalanine 55, threonine 55, tryptophan 250, valine 250; hypoxanthine 20, thymidine 5, adenosine 5, uridine 5, guanosine 5; 0.1 of selenium, 0.1 of chromium, 0.05 of cobalt, 0.05 of nickel, 0.1 of zinc, 0.1 of copper, 0.1 of manganese, 0.1 of barium, 0.05 of gallium, 0.1 of lithium, 0.1 of tin, 0.1 of iodine, 0.1 of vanadium, 0.05 of germanium, 0.05 of molybdenum, 0.1 of silicon, 0.1 of iron, 0.05 of rubidium, 0.05 of zirconium, 0.1 of cadmium and 0.1 of aluminum; 0.05 parts of biotin, 0.1 parts of D-calcium pantothenate, 0.05 parts of folic acid, 0.05 parts of nicotinamide, 0.05 parts of pyridoxine hydrochloride, 0.1 parts of riboflavin, 0.05 parts of thiamine hydrochloride, 120.1 parts of vitamin B, 20.1 parts of vitamin B and 0.1 parts of inositol; 500 parts of sodium chloride, 500 parts of potassium chloride, 55 parts of copper sulfate, 55 parts of ferric nitrate, 55 parts of ferrous sulfate, 55 parts of magnesium chloride, 250 parts of anhydrous disodium hydrogen phosphate, 250 parts of sodium dihydrogen phosphate, 250 parts of sodium selenite and 55 parts of zinc sulfate; cholesterol 5, lecithin 0.05, calcium chloride 5, linoleic acid 0.01, linolenic acid 0.01, lauric acid 0.1, palmitic acid 0.1, vitamin E0.1, prostaglandin 55, tween-805; glucose 8000, transferrin 55, insulin 55, bovine serum albumin 1000, soybean hydrolysate 1000; 1000 parts of sodium bicarbonate; phenol red 20; pluronic F68150; 0.02 parts of dexamethasone; amphotericin B500.
Preferably, the method for propagating the avian influenza virus by using the MDCK cell line comprises the following steps:
step one, mixing DMEM, F12 and RPMI1640 uniformly according to the proportion of 2-3: 1-2: 0.5-1, adding 10-12% FBS to the base culture medium, recovering and culturing adherent MDCK cells, after the cells grow to a compact monolayer, digesting the cells with 0.25% EDTA-pancreatin, and carrying out passage for 5-6 times; gradually reducing the proportion of DMEM-F12-RPMI1640 medium containing 10-12% FBS, and increasing the proportion of serum-free DMEM-F12-RPMI1640 medium to gradually reduce the serum content to 0%;
step two, digesting the well-grown MDCK cells which are adapted to serum-free culture by 0.25% EDTA-pancreatin, and then carrying out shake cultivation at the rotating speed of 30-50 r/min; after the growth rate of the cells is stable, carrying out passage, gradually enlarging culture, and gradually increasing the rotating speed of a shaking table until the cells are acclimatized into a stably-proliferated serum-free full-suspension culture type MDCK cell line;
step three, inoculating the domesticated MDCK cell line into a 3-5L bioreactor, wherein the inoculation density is 1-2 × 106cells/ml, culturing at 35-37 deg.C, pH 7.0-7.4, dissolved oxygen 30-50%, and amplification at 100-120r/min to 0.5-1 × 107completing primary cell culture at cells/ml;
inoculating the avian influenza H5N1 subtype virus solution propagated by the chick embryos to compact single-layer adherent MDCK cells, and culturing by using a virus maintenance solution; passage for 5-6 times; collecting culture supernatant when cytopathic effect reaches 80-90%, centrifuging at 2500-;
step five, taking the appropriate amount of the H5N1 subtype avian influenza virus cultured by adherence MDCK cells to proliferate according to MOI of 10-3-10-1Inoculating to 4.0-5.0 × 106cells/mL suspension MDCK cells, the culture conditions are 34-37 ℃, the pH7.2-7.4, the dissolved oxygen is 30% -50%, and the stirring speed is 100-.
Step six, culturing for 48-72h after inoculation, and separating and purifying virus particles when the cell viability is reduced to 40-70%.
Preferably, the method for propagating the avian influenza virus by using the MDCK cell line comprises the following steps:
step one, DMEM, F12 and RPMI1640 are uniformly mixed according to the ratio of 3: 1, the mixture is used as a basic culture medium, 10% FBS is added, adherent MDCK cells are recovered and cultured, after the cells grow to a compact monolayer, 0.25% EDTA-pancreatin is digested, and the cells are passaged for 5 times; gradually reducing the proportion of DMEM-F12-RPMI1640 medium containing 10% FBS, and increasing the proportion of serum-free DMEM-F12-RPMI1640 medium to gradually reduce the serum content from 10% to 0%;
step two, digesting the well-grown MDCK cells which are adapted to serum-free culture by 0.25% EDTA-pancreatin, and then carrying out shake cultivation at the rotating speed of 40 r/min; after the growth rate of the cells is stable, carrying out passage, gradually enlarging culture, and gradually increasing the rotating speed of a shaking table until the cells are acclimatized into a stably-proliferated serum-free full-suspension culture type MDCK cell line;
step three, inoculating the domesticated MDCK cell line into a 5L bioreactor, wherein the inoculation density is 2 × 106cells/ml, culturing at 37 deg.C, pH7.2, dissolved oxygen 40%, 110r/min, and amplifying to 1 × 107completing primary cell culture at cells/ml;
inoculating the avian influenza H5N1 subtype virus solution propagated by the chick embryos to compact single-layer adherent MDCK cells, and culturing by using a virus maintenance solution; passage 5 times; collecting culture supernatant when cytopathic effect reaches 90%, centrifuging at 3500r/min for 15min, removing precipitate, and collecting supernatant as seed poison for suspension MDCK cell culture;
step five, taking the appropriate amount of the H5N1 subtype avian influenza virus cultured by adherence MDCK cells to proliferate according to MOI of 10-2Inoculating to 5.0 × 106cells/mL suspension MDCK cells, the culture conditions are 34 ℃, pH7.3, dissolved oxygen is 40%, and the stirring speed is 110 r/min.
And step six, culturing for 60 hours after inoculation, separating supernatant and purifying virus particles when the cell viability is reduced to 50%.
It is another object of the present invention to provide a method for preparing an avian influenza virus vaccine, which comprises the steps of preparing a vaccine for avian influenza virus, and preparing a vaccine for avian influenza virus.
Preferably, the vaccine is one or more of recombinant vector vaccine, whole virus inactivated vaccine, attenuated live vaccine and nucleic acid vaccine.
Compared with the prior art, the method for proliferating the avian influenza virus by adopting the MDCK cell line has the following remarkable beneficial effects:
1. in the process of cell suspension culture and domestication, the invention adopts a method of gradually reducing serum, thereby avoiding the problem of cell inadaptation caused by sudden change of cell environment in a one-step method; further, by means of a shaking table system, cell suspension culture is realized, passage is stable, cell morphology is good, cell quality is high, and the method can be used for producing viruses.
2. The MDCK cells domesticated and obtained by the method can be efficiently proliferated in a bioreactor, the pollution probability is reduced, and the avian influenza H5N1 subtype viruses are cultured on the MDCK suspension cells, so that the sensitivity and the biological characteristics of the viruses can be maintained, the virus proliferation capacity is high, and the avian influenza virus strains can be efficiently proliferated.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
FIG. 1 shows the viability of MDCK cells in culture conditions with different serum contents during acclimation;
FIG. 2 is a diagram showing the state of MDCK cells after serum-free full-suspension acclimation;
FIG. 3 shows the effect of different virus inoculation times on virus titer under the virus inoculation amount determination conditions.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
Example 1
A method for propagating avian influenza virus by using MDCK cell line comprises the following steps:
step one, DMEM, F12 and RPMI1640 are uniformly mixed according to the ratio of 3: 2: 1, the mixture is used as a basic culture medium, 12% FBS is added, adherent MDCK cells are recovered and cultured, after the cells grow to a compact monolayer, 0.25% EDTA-pancreatin is digested, and the cells are passaged for 6 times; gradually reducing the proportion of DMEM-F12-RPMI1640 medium containing 12% FBS, and increasing the proportion of serum-free DMEM-F12-RPMI1640 medium to gradually reduce the serum content to 0%;
step two, digesting the well-grown MDCK cells which are adapted to serum-free culture by 0.25% EDTA-pancreatin, and then carrying out shake cultivation at the rotating speed of 50 r/min; after the growth rate of the cells is stable, carrying out passage, gradually enlarging culture, and gradually increasing the rotating speed of a shaking table until the cells are acclimatized into a stably-proliferated serum-free full-suspension culture type MDCK cell line;
step three, inoculating the domesticated MDCK cell line into a 5L bioreactor, wherein the inoculation density is 2 × 106cells/ml, culturing at 37 deg.C, pH7.4, dissolved oxygen 50%, 120r/min, and amplifying to 1 × 107completing primary cell culture at cells/ml;
inoculating the avian influenza H5N1 subtype virus solution propagated by the chick embryos to compact single-layer adherent MDCK cells, and culturing by using a virus maintenance solution; passage is carried out for 6 times; collecting culture supernatant when cytopathic effect reaches 85%, centrifuging at 3000r/min for 12min, removing precipitate, and collecting supernatant as seed poison for suspension MDCK cell culture;
step five, taking the appropriate amount of the H5N1 subtype avian influenza virus cultured by adherence MDCK cells to proliferate according to MOI of 10-2Inoculating to 5.0 × 106cells/mL suspension MDCK cells, the culture conditions are 37 ℃, pH7.4, dissolved oxygen 50%, and the stirring speed is 120 r/min.
Step six, culturing for 48-72h after inoculation, and separating and purifying virus particles when the cell viability is reduced to 60%.
Example 2
A method for propagating avian influenza virus by using MDCK cell line comprises the following steps:
step one, DMEM, F12 and RPMI1640 are uniformly mixed according to the ratio of 3: 1, the mixture is used as a basic culture medium, 10% FBS is added, adherent MDCK cells are recovered and cultured, after the cells grow to a compact monolayer, 0.25% EDTA-pancreatin is digested, and the cells are passaged for 5 times; gradually reducing the proportion of DMEM-F12-RPMI1640 medium containing 10% FBS, and increasing the proportion of serum-free DMEM-F12-RPMI1640 medium to gradually reduce the serum content from 10% to 0%;
step two, digesting the well-grown MDCK cells which are adapted to serum-free culture by 0.25% EDTA-pancreatin, and then carrying out shake cultivation at the rotating speed of 40 r/min; after the growth rate of the cells is stable, carrying out passage, gradually enlarging culture, and gradually increasing the rotating speed of a shaking table until the cells are acclimatized into a stably-proliferated serum-free full-suspension culture type MDCK cell line;
step three, inoculating the domesticated MDCK cell line into a 5L bioreactor, wherein the inoculation density is 2 × 106cells/ml, culturing at 37 deg.C, pH7.2, dissolved oxygen 40%, 110r/min, and amplifying to 1 × 107completing primary cell culture at cells/ml;
inoculating the avian influenza H5N1 subtype virus solution propagated by the chick embryos to compact single-layer adherent MDCK cells, and culturing by using a virus maintenance solution; passage 5 times; collecting culture supernatant when cytopathic effect reaches 90%, centrifuging at 3500r/min for 15min, removing precipitate, and collecting supernatant as seed poison for suspension MDCK cell culture;
step five, taking the appropriate amount of the H5N1 subtype avian influenza virus cultured by adherence MDCK cells to proliferate according to MOI of 10-3-10-1Inoculating to 5.0 × 106cells/mL suspension MDCK cells, the culture conditions are 34 ℃, pH7.3, dissolved oxygen is 40%, and the stirring speed is 110 r/min.
And step six, culturing for 60 hours after inoculation, separating supernatant and purifying virus particles when the cell viability is reduced to 50%.
Verification example 1
1 Material
1.1 cell strain and virus strain adherence MDCK cell, avian influenza H5N1 subtype virus strain
1.2 Virus maintaining culture solution prepared from Fetal Bovine Serum (FBS), DMEM/F12/RPMI1640 (3: 1), amino acids, choline chloride, vitamins, trace elements, etc
1.3 Instrument equipment cell shaker, 5L bioreactor, inverted microscope
2 Experimental methods and results
2.1 avian influenza subtype H5N1 virus propagation was performed using the method for propagating avian influenza virus using MDCK cell line described in example 2.
2.2 Activity of MDCK cells in the acclimation Process under different serum content culture conditions
In the process of cell serum-free domestication culture, from the initial 10% FBS-containing state to the complete serum-free state, the cells do not show obvious morphological difference, have good adherence, transparent cells and clear edges, are mostly triangular or irregular in shape, grow rapidly, have high activity and are stable for passage, and the figure is 1.
At the initial stage of serum-free suspension domestication, the cell activity is maintained at a lower level, the cells grow slowly and are aggregated seriously; after a period of adaptive culture, the cells gradually adapt to the serum-free suspension condition and proliferate, and the cell density is improved. After acclimation, the cells are round in shape, uniform in size, bright and singly suspended in a culture solution, the activity (the number of living cells/the total number of the cells) reaches more than 95%, and the cell states are shown in figure 2.
2.3 determination of optimum receiving dose MOI and optimum receiving time
2.3.1 seed-poisoning avian influenza subtype H5N1 Virus strains with different MOIs (10-3-10-1) The cells were inoculated into 3 bioreactors, and the number of cells in each bioreactor was kept the same, and the titer (HA titer, TCID50, EID50) was measured every 12h from 24h after inoculation until the cells were collected and measured (HA titer, TCID50, EID 50).
Wherein, HA titer is measured according to the method of pharmacopoeia of the people's republic of China, department III (Council of national pharmacopoeia, 2015); TCID50 measurement: serial 10-fold dilution of collected avian influenza virus in DMEM/F12/RPMI1640 medium-5、10-6、10-7And 10-84 dilutions are inoculated in a well-grown MDCK cell monolayer 96-well plate, 6 wells are inoculated in each dilution, and a blank control is set at the same time; culturing in a 5% CO2 incubator at 37 deg.C for 3-4 days, observing and recording cytopathic condition every 12h, and calculating TCID50 by Reed-Muench method; EID50 determination: diluting the collected avian influenza virus with PBS 10 times, and taking 10 times-5、10-6、10-7And 10-84 dilutions; inoculating 5 SPF (specific pathogen free) chick embryos of 10 days old, wherein each embryo is 0.1mL, and incubating for 96h at 37 ℃; the chick embryo liquid is harvested one by oneRespectively measuring HA titer; the HA titer is not less than 1: 16, the infection is judged, and the EID50 is calculated according to the Reed-Muench method.
As a result: with an MOI of 10-3When the virus inoculation amount is less, the virus infection amount is lower, so the propagation speed is reduced, and higher virus hemagglutination price and virus price cannot be achieved. With an MOI of 10-1When the virus is inoculated, the infection amount of the virus is too high, so that the infection is too fast, cells die early in the early stage of infection, and therefore higher virus hemagglutination price and virus price cannot be obtained. When the seed virus access amount MOI is 10-2And then, the virus multiplication is stable, the virus hemagglutination price and the virus price are high in the late virus harvest, and the HA highest value of the hemagglutination price is 1 in 72 hours: 1024, virus content up to 10 per 1mL8.93TCID50 with a viral content of up to 10 per 0.1mL7.87EID50, results are shown in Table 1.
TABLE 1 Effect of different MOI doses on the proliferation of avian influenza subtype H5N1 virus (HA, TCID50/mL, EID50/0.1mL)
2.3.2 determination of the optimum MOI inoculation dose to 10-2Then, the optimal harvest time was tested and analyzed under the condition of the inoculation amount, the HA titer, TCID50 and EID50 reached the highest 60h after inoculation, and the titer stabilized at this level without significant change at 72h after inoculation, and FIG. 3 illustrates that the optimal harvest time is 60h after inoculation.
According to the invention, the avian influenza H5N1 subtype virus is inoculated to serum-free full-suspension MDCK cells, three different virus receiving amounts are selected, and sampling is carried out at different time after virus receiving to monitor the virus price, so that the optimal virus receiving amount and the optimal virus receiving time are finally determined. MOI at Virus multiplicity of infection of 10-2The obtained virus has the highest toxin value, and the virus propagation can be better realized by proper virus inoculation amount. The invention provides reliable experimental basis for the large-scale production of the avian influenza vaccine.
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (7)
1. A method for propagating avian influenza virus by using MDCK cell line is characterized by comprising the following steps:
step one, mixing DMEM, F12 and RPMI1640 uniformly according to a certain proportion, taking the mixture as a basic culture medium, adding FBS, recovering and culturing adherent MDCK cells, and digesting and passaging after the cells grow to a compact monolayer; gradually reducing the FBS proportion to 0%;
step two, performing shake bed expansion culture on the well-grown serum-free cultured MDCK cells until the well-grown serum-free full-suspension culture MDCK cells become acclimatized into a stably-proliferated serum-free full-suspension culture MDCK cell line;
step three, inoculating the domesticated MDCK cell line into a bioreactor to complete primary cell culture;
inoculating the avian influenza H5N1 subtype virus solution propagated by the chick embryo to a compact single-layer adherent MDCK cell to obtain a seed virus for culturing the suspension MDCK cell;
step five, inoculating the avian influenza virus seed virus to a suspension MDCK cell for culture;
and step six, separating and purifying virus particles.
2. The method for propagating avian influenza virus by using MDCK cell line according to claim 1, which comprises the following steps:
step one, mixing DMEM, F12 and RPMI1640 uniformly according to the proportion of 2-3: 1-2: 0.5-1, adding 10-12% FBS to the base culture medium, recovering and culturing adherent MDCK cells, after the cells grow to a compact monolayer, digesting the cells with 0.25% EDTA-pancreatin, and carrying out passage for 5-6 times; gradually reducing the proportion of DMEM-F12-RPMI1640 medium containing 10-12% FBS, and increasing the proportion of serum-free DMEM-F12-RPMI1640 medium to gradually reduce the serum content to 0%;
step two, digesting the well-grown MDCK cells which are adapted to serum-free culture by 0.25% EDTA-pancreatin, and then carrying out shake cultivation at the rotating speed of 30-50 r/min; after the growth rate of the cells is stable, carrying out passage, gradually enlarging culture, and gradually increasing the rotating speed of a shaking table until the cells are acclimatized into a stably-proliferated serum-free full-suspension culture type MDCK cell line;
step three, inoculating the domesticated MDCK cell line into a 3-5L bioreactor, wherein the inoculation density is 1-2 × 106cells/ml, culturing at 35-37 deg.C, pH 7.0-7.4, dissolved oxygen 30-50%, and amplification at 100-120r/min to 0.5-1 × 107completing primary cell culture at cells/ml;
inoculating the avian influenza H5N1 subtype virus solution propagated by the chick embryos to compact single-layer adherent MDCK cells, and culturing by using a virus maintenance solution; passage for 5-6 times; collecting culture supernatant when cytopathic effect reaches 80-90%, centrifuging at 2500-;
step five, taking the appropriate amount of the H5N1 subtype avian influenza virus cultured by adherence MDCK cells to proliferate according to MOI of 10-3-10-1Inoculating to 4.0-5.0 × 106cells/mL suspension MDCK cells, the culture conditions are 34-37 ℃, the pH7.2-7.4, the dissolved oxygen is 30% -50%, and the stirring speed is 100-.
Step six, culturing for 48-72h after inoculation, and separating and purifying virus particles when the cell viability is reduced to 40-70%.
3. The method for propagating avian influenza virus by using MDCK cell line as claimed in claim 2, wherein the virus maintenance solution is composed of the following components in mg/L: serine 250, arginine 250, leucine 55, isoleucine 250, alanine 2, tyrosine 55, asparagine 25, aspartic acid 25, cysteine hydrochloride 55, glutamic acid 50, glutamine 50, histidine 250, lysine 250, phenylalanine 55, threonine 55, tryptophan 250, valine 250; hypoxanthine 20, thymidine 5, adenosine 5, uridine 5, guanosine 5; 0.1 of selenium, 0.1 of chromium, 0.05 of cobalt, 0.05 of nickel, 0.1 of zinc, 0.1 of copper, 0.1 of manganese, 0.1 of barium, 0.05 of gallium, 0.1 of lithium, 0.1 of tin, 0.1 of iodine, 0.1 of vanadium, 0.05 of germanium, 0.05 of molybdenum, 0.1 of silicon, 0.1 of iron, 0.05 of rubidium, 0.05 of zirconium, 0.1 of cadmium and 0.1 of aluminum; 0.05 parts of biotin, 0.1 parts of D-calcium pantothenate, 0.05 parts of folic acid, 0.05 parts of nicotinamide, 0.05 parts of pyridoxine hydrochloride, 0.1 parts of riboflavin, 0.05 parts of thiamine hydrochloride, 120.1 parts of vitamin B, 20.1 parts of vitamin B and 0.1 parts of inositol; 500 parts of sodium chloride, 500 parts of potassium chloride, 55 parts of copper sulfate, 55 parts of ferric nitrate, 55 parts of ferrous sulfate, 55 parts of magnesium chloride, 250 parts of anhydrous disodium hydrogen phosphate, 250 parts of sodium dihydrogen phosphate, 250 parts of sodium selenite and 55 parts of zinc sulfate; cholesterol 5, lecithin 0.05, calcium chloride 5, linoleic acid 0.01, linolenic acid 0.01, lauric acid 0.1, palmitic acid 0.1, vitamin E0.1, prostaglandin 55, tween-805; glucose 8000, transferrin 55, insulin 55, bovine serum albumin 1000, soybean hydrolysate 1000; 1000 parts of sodium bicarbonate; phenol red 20; pluronic F68150; 0.02 parts of dexamethasone; amphotericin B500.
4. The method for propagating avian influenza virus using MDCK cell line according to any of claims 1-3, wherein the inoculation amount MOI in step five is 10-2And the culture time after the inoculation in the step six is 60 hours.
5. The method for propagating avian influenza virus using MDCK cell line according to any of claims 1-3, wherein the culture conditions in step five are 34 ℃, pH7.3, dissolved oxygen 40%, and stirring speed 110 r/min.
6. Use of a fully suspension cultured MDCK cell line obtained according to the method of any one of claims 1-5 for the preparation of an avian influenza virus vaccine.
7. The use according to claim 6, wherein the vaccine is one or more of recombinant vector vaccine, whole virus inactivated vaccine, attenuated live vaccine and nucleic acid vaccine.
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