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Full suspension culture method of duck hepatitis virus

A technology of duck hepatitis virus and culture method, which is applied in the field of veterinary biological products, can solve the problems of no cell-derived duck hepatitis virus vaccine, decreased culture titer of duck hepatitis virus, failure to meet production requirements, etc., to achieve large-scale cultivation, The effect of simplifying the product separation and purification process and improving the utilization rate of equipment

Inactive Publication Date: 2019-02-01
ZHAOQING INST OF BIOTECHNOLOGY CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported cells used for duck hepatitis virus culture mainly include primary cells such as chicken or duck embryo fibroblasts, duck embryo kidney cells, etc., but because the preparation of primary cells is essentially carried out by taking chicken or duck embryo embryos Production, the procedure is more complicated on the contrary, and adopt above-mentioned cells to carry out the culture titer of duck hepatitis virus to reduce greatly, generally can only reach 10 3.0 -10 5.0 ELD 50 / mL, unable to meet the production requirements, because this bottleneck cannot be broken through, so the use of cells to culture duck hepatitis virus has been in the research stage, and there is no report of cell-derived duck hepatitis virus vaccine on the market.

Method used

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  • Full suspension culture method of duck hepatitis virus
  • Full suspension culture method of duck hepatitis virus
  • Full suspension culture method of duck hepatitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Effects of different doses of inoculation and culture time on the proliferation of duck hepatitis virus in passaged cells derived from duck embryo cells

[0032] The sources of materials used in Embodiment 1 of the present invention are as follows:

[0033] 1. Virus: duck hepatitis virus attenuated strain GD75, identified, kept and supplied by Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd.

[0034] 2. Cells: The whole suspension passage cell line is a passage cell line derived from duck embryo cells.

[0035] 3. Culture medium: the name is medium EBLM004, containing 10mg / L alanine, 10mg / L arginine, 10mg / L cysteine, 10mg / L tyrosine, 10mg / L chromosine amino acid, 10mg / L valine, 10mg / L leucine, 1mg / L vitamin C, 1mg / L biotin, 1mg / L folic acid, 1mg / L choline chloride, 1mg / L inositol, 1mg / L Niacinamide, 1mg / L Pyridoxine Hydrochloride, 50mg / L Potassium Chloride, 50mg / L Sodium Chloride, 50mg / L Disodium Hydrogen Phosphate, 50mg / L Sodium Bicarbonate, 1000mg / L...

Embodiment 2

[0046] Example 2: ELD of Duck Hepatitis Virus at Different Harvesting Time in Duck Embryo Cell-Derived Passage Cell Line Culture 50 Compare

[0047] The source of materials used in Embodiment 2 of the present invention is as follows:

[0048] 1. Virus: Adapted strain of duck hepatitis virus (GD75 strain) in passage cells derived from duck embryo cells.

[0049] 2. Cells: The whole suspension passage cell line is a passage cell line derived from duck embryo cells.

[0050] 3. Culture medium: the name is medium EBLM004, containing 10mg / L alanine, 10mg / L arginine, 10mg / L cysteine, 10mg / L tyrosine, 10mg / L chromosine amino acid, 10mg / L valine, 10mg / L leucine, 1mg / L vitamin C, 1mg / L biotin, 1mg / L folic acid, 1mg / L choline chloride, 1mg / L inositol, 1mg / L Niacinamide, 1mg / L Pyridoxine Hydrochloride, 50mg / L Potassium Chloride, 50mg / L Sodium Chloride, 50mg / L Disodium Hydrogen Phosphate, 50mg / L Sodium Bicarbonate, 1000mg / L Glucose, 0.1mg / L manganese sulfate, 0.01mg / L ferric nitrate,...

Embodiment 3

[0058] Example 3: Stable passage of duck hepatitis virus in passage cells derived from duck embryo cells

[0059] The present invention implements 3 sources of materials used as follows:

[0060] 1. Virus: ELD harvested from previous generation 50 The highest duck hepatitis virus was used as the seed virus.

[0061] 2. Cells: Passage cells derived from duck embryo cells of the whole suspension passage cell line.

[0062] 3. Culture medium: the name is medium EBLM004, containing 10mg / L alanine, 10mg / L arginine, 10mg / L cysteine, 10mg / L tyrosine, 10mg / L chromosine amino acid, 10mg / L valine, 10mg / L leucine, 1mg / L vitamin C, 1mg / L biotin, 1mg / L folic acid, 1mg / L choline chloride, 1mg / L inositol, 1mg / L Niacinamide, 1mg / L Pyridoxine Hydrochloride, 50mg / L Potassium Chloride, 50mg / L Sodium Chloride, 50mg / L Disodium Hydrogen Phosphate, 50mg / L Sodium Bicarbonate, 1000mg / L Glucose, 0.1mg / L manganese sulfate, 0.01mg / L ferric nitrate, 0.1mg / L insulin, 0.5mg / L EGF, 1mg / LbFGF.

[0063] ...

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Abstract

The invention discloses a full suspension culture method of duck hepatitis virus, wherein the method comprises the following steps: step 1, carrying out resuscitation and subculture of subcultured cell lines derived from duck embryo cells; step 2, adopting a second-order culture method, inoculating the subcultured cells derived from the duck embryo cells subjected to full suspension culture with the duck hepatitis virus, and carrying out virus large-scale culture; and step 3, after inoculating with the virus, taking samples every other 12 h and measuring the virus ELD50, harvesting the virus and preserving when the virus ELD50 reaches the maximum, and thus obtaining the duck hepatitis virus after culture. The full suspension culture method improves the scale and efficiency of virus culture, conforms to the tendency of future vaccine production, has broad application prospects and contains good economic benefits.

Description

technical field [0001] The invention relates to a full-suspension culture method of duck hepatitis virus, in particular to a method for full-suspension culture of duck hepatitis virus with passaged cell lines, and belongs to the technical field of veterinary biological products. Background technique [0002] In recent years, duck viral hepatitis has been widely prevalent in waterfowl breeding areas in China, posing a serious threat to the healthy development of the duck industry. Duck viral hepatitis is caused by duck hepatitis virus (DHV), which mainly causes the disease of ducklings under 3 weeks of age, and the fatality rate can reach 100%. There are three serotypes of duck hepatitis virus reported so far: type Ⅰ, type Ⅱ and type Ⅲ. Type Ⅰ and type Ⅲ duck hepatitis viruses have been found in China, among which type Ⅰ is the most prevalent. At present, there is no specific drug to treat the disease. Vaccination against duck hepatitis is the most economical and effective ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/073C12R1/93
CPCC12N5/0603C12N7/00C12N2770/32351
Inventor 温良海陈瑞爱蔡仕君叶俊贤谭银娟
Owner ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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