A kind of preparation method of the avian influenza virus grown in the mdck cell growth of serum-free full suspension culture and the avian influenza virus obtained

An avian influenza virus, serum-free culture medium technology, applied in the field of avian influenza virus, can solve the problems of increasing the complexity of the process, production time and cost, difficulty in large-scale cultivation, etc. Conducive to inoculation and culture, high activity

Active Publication Date: 2019-10-15
ZHAOQING DAHUANONG BIOLOGIC PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 of cells 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 of

Method used

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  • A kind of preparation method of the avian influenza virus grown in the mdck cell growth of serum-free full suspension culture and the avian influenza virus obtained
  • A kind of preparation method of the avian influenza virus grown in the mdck cell growth of serum-free full suspension culture and the avian influenza virus obtained
  • A kind of preparation method of the avian influenza virus grown in the mdck cell growth of serum-free full suspension culture and the avian influenza virus obtained

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Experimental program
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preparation example Construction

[0090] Three, a kind of preparation method of the avian influenza virus that adapts to the MDCK cell line growth of serum-free full suspension culture, may further comprise the steps:

[0091] 1) Prepare the MDCK cells adapted to the serum-free full suspension culture to be inoculated: put the MDCK cells into a shake flask, at a rotation speed of 130 rpm, at a temperature of 37° C., and inject CO with a concentration of 5%. 2 cultured in an incubator under certain conditions; then samples were taken every 12 hours for cell counting; when MDCK cells were in the logarithmic growth phase, diluted with fresh serum-free medium until the density of MDCK cells was 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 adapted to serum-free full suspension culture to be inoculated;

[0092] 2) Prepare virus seeds: prepare chicken embryo-derived H9N2 subtype avian influenza virus; the virus content of avia...

Embodiment 1

[0100] The serum-free medium includes the following components in terms of concentration:

[0101] Basal Metabolic Nutrients:

[0102]

[0103] Nucleotides:

[0104]

[0105] Vitamins:

[0106]

[0107] Inorganic salt:

[0108]

[0109]

[0110] Shear protector:

[0111] Block polyether F68 1600mg / L;

[0112] Anti-cell clumping agent:

[0113] Dextran sulfate 50mg / L;

[0114] pH buffer:

[0115] Sodium bicarbonate 2200mg / L;

[0116] pH indicator:

[0117] Phenol red 8mg / L;

[0118] Influenza virus proliferation promoter:

[0119]

[0120] Other additions:

[0121]

[0122] 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 the serum-free medium is obtained after constant volume.

Embodiment 2

[0124] The serum-free medium includes the following components in terms of concentration:

[0125] Basal Metabolic Nutrients:

[0126]

[0127]

[0128] Nucleotides:

[0129]

[0130] Vitamins:

[0131]

[0132]

[0133] Inorganic salt:

[0134]

[0135] Shear protector:

[0136] Block polyether F68 1000mg / L;

[0137] Anti-cell clumping agent:

[0138] Dextran sulfate 25mg / L;

[0139] pH buffer:

[0140] Sodium bicarbonate 2200mg / L;

[0141] pH indicator:

[0142] Phenol red 8mg / L;

[0143] Influenza virus proliferation promoter:

[0144]

[0145]

[0146] Other additions:

[0147]

[0148] 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 the 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 cultivation and domestication, in particular to a method for preparing an avian influenza virus adapted to grow in a serum-free full suspension cultured MDCK cell line and an 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 carrier, the cost of the vaccine is relatively high, and there is no way to deal with the acute and large-scale avian influenza epidemic. 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 of cells 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/02C12N5/071C12N5/02
Inventor 陈瑞爱赖汉漳谭文松詹烜子刘旭平麦康聪刘玉鹏汤钦盘伟岚陈华坚王小芬陈培军许冬蕾
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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