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Method for proliferating avian influenza virus on MDCK full suspension cells and application thereof

An avian influenza virus, full suspension technology, applied in the direction of viruses, animal cells, viruses/phages, etc., can solve the problems of large amount of seed virus access, long domestication time, long virus domestication cycle, etc., to shorten the production cycle, seed virus The effect of variant risk reduction

Active Publication Date: 2018-11-06
吉林冠界生物技术有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0002] The existing method for the propagation of recombinant avian influenza virus on MDCK suspension cells mainly adopts chicken embryo seed virus to carry out 5-10 generations of passage domestication on adherent MDCK cells, so that the virus is gradually adapted to multiply on adherent cells. Then inoculate the adherent virus that has been acclimatized and adapted on the adherent cells to the suspension cells and then carry out 1-3 generations of subculture and domestication. Only the viruses adapted to the suspension cells can be put into large-scale production for antigen production. The entire seed virus domestication The process requires a lot of manpower and material resources, and the domestication time is also long (3-6 months). Even after the domestication is completed, the amount of inoculated poison will be relatively large (0.1%-0.3%)
Furthermore, since the avian influenza virus has 16 HA subtypes and 9 NA subtypes, it is relatively easy to mutate, and its immunogenicity may undergo irreversible changes after continuous passage on cells.
[0003] To sum up, the currently used recombinant avian influenza virus propagation method on MDCK suspension cells is not only relatively complicated, but also the virus domestication cycle will be relatively long, which is not conducive to the timely and stable supply of vaccines during influenza pandemics. It is also difficult to guarantee the immune effect of continuous passage on cells

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  • Method for proliferating avian influenza virus on MDCK full suspension cells and application thereof
  • Method for proliferating avian influenza virus on MDCK full suspension cells and application thereof
  • Method for proliferating avian influenza virus on MDCK full suspension cells and application thereof

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preparation example Construction

[0034] The preparation method of the culture medium described in any one of the foregoing includes: mixing all the culture medium components together to form dry culture medium powder and then dispersing in a solvent.

[0035] Preferably, at the time of preparation, the major components of the medium are refined and mixed together to form a dry medium powder, and the minor components are separately formulated into a minor component liquid.

[0036] Preferably, in the above-mentioned preparation method, the method further includes pH adjustment and filter sterilization.

[0037] Preferably, the method as described above, the method also includes:

[0038] Viral antigens are isolated from the virions and purified and / or inactivated.

[0039] According to one aspect of the present invention, the present invention also relates to the application of the aforementioned method in the preparation of avian influenza virus vaccine.

[0040] Preferably, in the above-mentioned applicati...

Embodiment 1

[0043] A serum-free medium adapted to the full suspension culture of MDCK cells, calculated as 1 liter of solution after dissolving with ultrapure water, said medium includes the following components:

[0044] Biotin 1 x 10 -8 M, Calcium Chloride 5×10 -3 M, copper sulfate 2.8×10 -9 M, Cyanocobalamin 1×10 -7 M, D-calcium pantothenate 2×10 -5 M, D-glucose 2.0×10 -2 M, ferrous sulfate 2×10 -6 M, folic acid 0.5×10 -4 M. Glutathione 9.5×10 -7 M. Hydrocortisone 3×10 -8 M, Hypoxanthine 1×10 -5 M. Inositol 10×10 -5 M, ferric nitrate 0.3×10 -7 M, L-alanine 20×10 -5 M, L-arginine 20×10 -4 M, L-asparagine 1×10 -5 M, L-aspartic acid 20×10 -5 M, L-cysteine ​​0.1×10 - 4 M, L-cystine 10×10 -4 M, L-glutamic acid 1×10 -5 M, L-Glutamine 10×10 -3 M, Glycine 1×10 -4 M, L-histidine 10×10 -4 M, L-isoleucine 0.5×10 -4 M, L-Leucine 10×10 -4 M, L-lysine 1×10 -4 M, L-methionine 10×10 -4 M, L-phenylalanine 1×10 -4M, L-proline 10×10 -4 M, L-serine 1×10 -4 M, L-Threonine 20×10 ...

Embodiment 2

[0047] A serum-free medium adapted to the full suspension culture of MDCK cells, calculated as 1 liter of solution after dissolving with ultrapure water, said medium includes the following components:

[0048] Biotin 3 x 10 -8 M. Calcium chloride 2×10 -3 M, copper sulfate 7.8×10 -9 M, Cyanocobalamin 3×10 -7 M, D-calcium pantothenate 5×10 -5 M, D-glucose 1.8×10 -2 M, ferrous sulfate 5×10 -6 M, folic acid 1×10 -4 M. Glutathione 6.5×10 -7 M. Hydrocortisone 5×10 -8 M, Hypoxanthine 3×10 -5 M. Inositol 7×10 -5 M, ferric nitrate 1.2×10 -7 M, L-alanine 5×10 -5 M, L-arginine 7×10 -4 M, L-asparagine 5×10 -5 M, L-aspartic acid 5×10 -5 M, L-cysteine ​​1×10 -4 M, L-cystine 1×10 -4 M, L-glutamic acid 5×10 -5 M, L-Glutamine 2.5×10 -3 M. Glycine 2.5×10 -4 M, L-histidine 1.5×10 -4 M, L-isoleucine 4.2×10 -4 M, L-leucine 4.5×10 -4 M, L-lysine 5×10 -4 M, L-methionine 1.2×10 - 4 M, L-phenylalanine 2.2×10 -4 M, L-proline 1.5×10 -4 M, L-serine 2.5×10 -4 M, L-threonine 4.5...

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Abstract

The invention relates to a method for proliferating avian influenza virus on MDCK full suspension cells and application thereof. The method comprises the steps of: inoculating, chicken embryo avian influenza virus according to MOI equal to 0.001 to 0.0001 when MDCK cells are cultured to 7.0*10<6> to 1.0*10<7> cells / mL in a full suspension manner; cultivating at pH value of 6.9 to 7.3, dissolved oxygen of 30 to 60%, and temperature of 32 to 37 DEG C after virus inoculation, and separating and purifying the virions, when the virus reaches a sufficiently high titer. The method, due to that fact that the seed virus does not need circulation, shortens the production cycle and greatly reduces the risk of virus variation. The HA of recombinant avian influenza virus cultivated by the method can beup to 1 to 1024, the virus content per 1 ml is more than or equal to 10<8.37>TCID<50>,the virus content per 0.1 ml is more than or equal to 10<8.17>EID<50>,, and the inoculation amount of the seed virus is reduced by 10 times or more.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for propagating avian influenza virus on MDCK full-suspension cells and its application. Background technique [0002] The existing method for the propagation of recombinant avian influenza virus on MDCK suspension cells mainly adopts chicken embryo seed virus to carry out 5-10 generations of passage domestication on adherent MDCK cells, so that the virus is gradually adapted to multiply on adherent cells. Then inoculate the adherent virus that has been acclimatized and adapted on the adherent cells to the suspension cells and then carry out 1-3 generations of subculture and domestication. Only the viruses adapted to the suspension cells can be put into large-scale production for antigen production. The entire seed virus domestication The process needs to consume a large amount of manpower and material resources, and the domestication time is also long (3-6 months)....

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N5/071C12N5/02
CPCC12N5/0686C12N7/00C12N2500/12C12N2500/14C12N2500/24C12N2500/25C12N2500/32C12N2500/34C12N2500/38C12N2500/40C12N2500/44C12N2500/46C12N2501/395C12N2760/16051
Inventor 陈宏王博赵博崔凯黄琳沈玥李莉杜鑫张丽娜金燕斌朱长动
Owner 吉林冠界生物技术有限公司
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