Preparation method of avian influenza virus growing in serum-free full-suspended cultured MDCK cells and obtained avian influenza virus

A technology of avian influenza virus and serum-free medium, applied in the field of avian influenza virus, can solve the problems of difficult large-scale cultivation, increase the complexity of the process, production time and cost, and achieve the improvement of virus content and HA hemagglutination titer, Conducive to inoculation and cultivation, short acclimatization time

Active Publication Date: 2016-10-12
ZHAOQING DAHUANONG BIOLOGIC PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a small number of manufacturers have adopted the adherent MDCK cell culture method to prepare H5 avian influenza vaccine. However, in the single-cell layer culture system, the proliferation o...

Method used

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  • Preparation method of avian influenza virus growing in serum-free full-suspended cultured MDCK cells and obtained avian influenza virus
  • Preparation method of avian influenza virus growing in serum-free full-suspended cultured MDCK cells and obtained avian influenza virus
  • Preparation method of avian influenza virus growing in serum-free full-suspended cultured MDCK cells and obtained avian influenza virus

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Experimental program
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preparation example Construction

[0090] 3. A preparation method of avian influenza virus adapted to grow in MDCK cell line cultured in serum-free full suspension, including the following steps:

[0091] 1) Prepare MDCK cells to be inoculated for serum-free full suspension culture: Put MDCK cells into a shaker flask at a rotation speed of 130 rpm, a temperature of 37°C, and a 5% CO concentration 2 Placed in an incubator under the conditions of, and then sampled every 12h to count the cells; when MDCK cells are in the logarithmic growth phase, dilute them with fresh serum-free medium to a density of 0.5×10 6 cells / mL, and use this as the initial density of MDCK cells, and then culture the MDCK cells for 72 hours to obtain MDCK cells suitable for serum-free full suspension culture to be inoculated;

[0092] 2) Preparation of virus seed: prepare H9N2 subtype avian influenza virus from chicken embryo; the viral content of avian influenza virus is ≥10 6.0 EID 50 / 0.1mL;

[0093] 3) F1 generation virus domestication: inocul...

Embodiment 1

[0099] The serum-free medium includes the following components in terms of concentration:

[0100] Basic metabolic nutrients:

[0101]

[0102] Nucleotide:

[0103]

[0104] Vitamins:

[0105]

[0106] Inorganic salt:

[0107]

[0108]

[0109] Shear protection agent:

[0110] Block polyether F68 1600mg / L;

[0111] Anti-cell clumping agent:

[0112] Dextran sulfate 50mg / L;

[0113] PH buffer:

[0114] Sodium bicarbonate 2200mg / L;

[0115] PH indicator:

[0116] Phenol red 8mg / L;

[0117] Influenza virus proliferation promoter:

[0118]

[0119] Other additives:

[0120]

[0121] The raw materials are mixed and ground into fine powder, and then the obtained fine powder is dissolved in a solvent at 10-30°C to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.5, and a serum-free medium is obtained after constant volume.

Embodiment 2

[0123] The serum-free medium includes the following components in terms of concentration:

[0124] Basic metabolic nutrients:

[0125]

[0126]

[0127] Nucleotide:

[0128]

[0129] Vitamins:

[0130]

[0131]

[0132] Inorganic salt:

[0133]

[0134] Shear protection agent:

[0135] Block polyether F68 1000mg / L;

[0136] Anti-cell clumping agent:

[0137] Dextran sulfate 25mg / L;

[0138] PH buffer:

[0139] Sodium bicarbonate 2200mg / L;

[0140] PH indicator:

[0141] Phenol red 8mg / L;

[0142] Influenza virus proliferation promoter:

[0143]

[0144]

[0145] Other additives:

[0146]

[0147] The raw materials are mixed and ground into fine powder, and then the obtained fine powder is dissolved in a solvent at 10-30° C. to obtain a mixed solution; the pH of the mixed solution is adjusted to 6.4, and a serum-free medium is obtained after constant volume.

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Abstract

The invention discloses a preparation method of an avian influenza virus suitable for growing in a serum-free full-suspended cultured MDCK cell line and the avian influenza virus obtained through the method. The preparation method comprises the following steps of 1 preparation of the MDCK cells to be inoculated; 2 virus seed preparation, wherein a chick embryo source avian influenza virus is prepared; 3 F1 generation virus domestication; 4 F2 generation virus domestication, wherein a supernatant sample retained at the time point when the blood clotting titer of the avian influenza virus obtained in the F1 generation is highest is taken, and the step 3 is repeated; 5 F3 generation virus domestication, wherein the F3 generation virus culturing temperature is 35 DEG C; 6 F4 generation-F10 generation virus domestication, wherein the step 5 is repeated, and the domesticated avian influenza virus is obtained. According to the preparation method, through a domestication method, the avian influenza virus is directly domesticated to completely adapt to be efficiently reproduced on the serum-free full-suspended cultured MDCK cells from the mode of being cultured by a chick embryo, the domestication efficiency is high, the avian influenza virus can be efficiently infected and copied in the MDCK cells, and the virus characteristic is stable.

Description

Technical field [0001] The invention relates to the field of virus culture and domestication, in particular to a preparation method of avian influenza virus adapted to grow in a MDCK cell line cultured in serum-free full suspension and avian influenza virus obtained by the method. Background technique [0002] The H9 subtype avian influenza inactivated vaccines currently produced in my country all use chicken embryos as the virus growth vector. The cost of the vaccine is high and there is no way to deal with the acute large-scale avian influenza epidemic. At present, a few manufacturers have adopted the adherent MDCK cell culture method to prepare H5 avian influenza vaccine. However, in the monolayer culture system, cell proliferation is limited by the surface area of ​​the substrate, which makes it difficult to achieve large-scale culture, and the digestion process also increases the process Complexity, production time and cost. Serum-free suspension culture can break through t...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02C12N5/071C12N5/02
Inventor 陈瑞爱赖汉漳刘玉鹏詹烜子刘旭平麦康聪汤钦盘伟岚许东蕾陈华坚
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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