A method for producing porcine transmissible gastroenteritis virus using a bioreactor

A bioreactor and infectious technology, applied in the field of using bioreactors to produce swine transmissible gastroenteritis virus, can solve the problems of high production cost, difficult production, high labor intensity, etc., to improve vaccine quality and output, reduce The effect of production cost and short production cycle

Active Publication Date: 2016-01-13
兆丰华生物科技(南京)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional process is labor-intensive, time-consuming, low in efficiency, and high in production cost; it is easy to be polluted by the environment; there are large differences between different production batches or between different bottles of the same production batch; it is difficult to expand production; it is easy to be contaminated by bacteria or other viruses The hidden dangers of vaccine quality, involving biosafety and public health issues

Method used

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  • A method for producing porcine transmissible gastroenteritis virus using a bioreactor
  • A method for producing porcine transmissible gastroenteritis virus using a bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] (1) Choose a bioreactor as the means of cultivation: 7L Swiss Lambda bioreactor.

[0031] (2) Select the microcarrier as the carrier for cell attachment and growth: microcarrier Cytodex2.

[0032] (3) Cleaning and sterilization methods of microcarriers: 1) Weigh 10 g / L of Cytodex2 microcarriers and soak in 2LPBS solution for 3 hours. 2) Wash 3 times with 2LPBS each time. 3) Add 2LPBS solution to soak the microcarriers, and steam sterilize at 121° C. for 30 minutes.

[0033] (4) Select ST cells as cells for making seedlings.

[0034] (5) Passaging and culturing of cells for seedling preparation: the above-mentioned cells were digested and passaged by EDTA-trypsin (Hank'S solution containing 0.25% trypsin and 0.02% EDTA), and then prepared with 95% MEM solution, 5% calf serum, 200IU / mL of penicillin sodium and streptomycin sulfate, the pH value of the cell growth medium was adjusted to 7.2, and the culture temperature was 37°C. When a good monolayer is formed, it is ...

Embodiment 2

[0040] (1) Choose a bioreactor as the means of cultivation: 7L Swiss Lambda bioreactor.

[0041] (2) Select the microcarrier as the carrier for cell attachment and growth: microcarrier Cytodex3.

[0042] (3) Cleaning and sterilization methods of microcarriers: 1) Weigh Cytodex3 microcarriers at 6 g / L and soak in 2 LPBS solution for 3 hours. 2) Wash 3 times with 2LPBS each time. 3) Add 2LPBS solution to soak the microcarriers, and steam sterilize at 121° C. for 30 minutes.

[0043] (4) Select vero cells as cells for seedling production.

[0044] (5) Passaging and culturing of cells for seedling preparation: the above-mentioned cells were digested and passaged by EDTA-trypsin (Hank'S solution containing 0.25% trypsin and 0.02% EDTA), and then prepared with 96% MEM solution, 4% calf serum, 200IU / mL of penicillin sodium and streptomycin sulfate, the pH value of the cell growth medium was adjusted to 7.2, and the culture temperature was 37°C. When a good monolayer is formed, i...

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Abstract

The invention provides a production method of porcine transmissible gastroenteritis virus by utilizing a bioreactor. The method comprises the following steps: 1) preparing monolayer subculture cells; 2) preparing virus seed for porcine transmissible gastroenteritis virus production; 3) preparing a cell suspension from the monolayer subculture cells prepared in the step 1), and inoculating into a bioreactor to carry out adsorption culture on the subculture cells in a microcarrier in the bioreactor; 4) inoculating the virus seed prepared in the step 2) at the inoculation amount of 2 to 5 percent when the subculture cells grow to 80-90 percent of the microcarrier, the empty bead rate is lower than 5 percent, the full bead rate is more than 80 percent and the cell count is over 3-5*10<6> per mL, and performing virus adsorption culture; and 5) harvesting virus fluid when over 80 percent of the subculture cells on the microcarrier have pathological changes. The method can be used for solving the problems of low production efficiency, unstable product quality and low virus titer, so that the unit vulture titer of the virus can be improved by 5 to 10 times, the quality and yield of vaccine can be comprehensively improved, and the safety of vaccine is improved.

Description

technical field [0001] The invention relates to a method for producing a virus, in particular to a method for producing porcine transmissible gastroenteritis virus, and more specifically to a method for producing porcine transmissible gastroenteritis virus by utilizing a bioreactor. Background technique [0002] Porcine transmissible gastroenteritis (Porcine Transmissible Gastroenteritis, TGE) is caused by porcine transmissible gastroenteritis virus (Porcine Transmissible Gastroenteritis virus, TGEV) of Coronaviridae (Coronaviridae) and Coronavirus (Coronavirus), characterized by pig vomiting, diarrhea, and dehydration An infectious disease that has a high lethality rate for newborn piglets, usually 100%. Although pigs of different ages are susceptible to the virus, pigs older than 5 weeks have a low mortality rate after infection. After Doyle reported that the disease first occurred in the United States in 1945, many countries in the world have reported porcine transmissib...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 于萍萍王贵华侯艳红
Owner 兆丰华生物科技(南京)有限公司
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