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Method for culturing porcine epidemic diarrhea virus

A technology of porcine epidemic diarrhea and culture methods, applied in the direction of microorganism-based methods, viruses/bacteriophages, biochemical equipment and methods, etc., which can solve the problems of difficult cultivation of wild strains

Active Publication Date: 2015-05-13
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Many domestic and foreign literature reports pointed out that the clinically isolated porcine epidemic diarrhea virus can be infected with a variety of cells in vitro, such as VERO cells, BHK cells, etc., but it is difficult for the clinically isolated wild strain of porcine epidemic diarrhea virus to After cultivation, half of the tissue cell infection rate (TCID50) was basically less than measured, which caused a huge problem for the expanded production of the virus and the determination of the virus amount. Therefore, how to improve the infection efficiency of the virus to cells and adapt it to large-scale culture in vitro is a must. The difficulty of culturing the virus in vitro

Method used

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  • Method for culturing porcine epidemic diarrhea virus
  • Method for culturing porcine epidemic diarrhea virus
  • Method for culturing porcine epidemic diarrhea virus

Examples

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Effect test

Embodiment 1

[0044] (1) Preparation of DMEM medium: Dissolve DMEM dry powder in deionized water, stir until completely dissolved, adjust the pH value to 7.2, and sterilize and filter with a sterile filter membrane of ≤0.22 μm, and place it in a temperature of ≤4°C Refrigerator for standby use;

[0045] (2) adding the fetal bovine serum that accounts for 1-8% of the volume of the DMEM medium in the above-mentioned DMEM medium, and placing the DMEM medium in the culture vessel to carry out the cultivation of Vero monolayer cells;

[0046] (3) Discard the DMEM medium in the culture vessel where the monolayer cells are formed, and wash 2-3 times with a washing solution; the washing solution is sterile deionized water.

[0047] (4) Discard the washing solution, and add porcine trypsin, cell affinity agent and DMEM medium in step (1) to the culture vessel formed with monolayer cells, wherein the consumption of porcine trypsin is the volume consumption of DMEM medium 0.3% of DMEM, and the dosage o...

Embodiment 2

[0050] (1) Preparation of DMEM medium: Dissolve DMEM dry powder in deionized water, stir until completely dissolved, adjust the pH value to 6.8, and sterilize and filter with a sterile filter membrane of ≤0.22 μm, and place it in a temperature of ≤4°C Refrigerator for standby use;

[0051] (2) adding the fetal bovine serum that accounts for 1-8% of the volume of the DMEM medium in the above-mentioned DMEM medium, and placing the DMEM medium in the culture vessel to carry out the cultivation of Vero monolayer cells;

[0052] (3) Discard the DMEM medium in the culture vessel where the monolayer cells are formed, and wash 2-3 times with a washing solution; the washing solution is sterile deionized water.

[0053] (4) Discard the washing solution, and add porcine trypsin, cell affinity agent and DMEM medium in step (1) to the culture vessel formed with monolayer cells, wherein the consumption of porcine trypsin is the volume consumption of DMEM medium 0.1% of DMEM, and the dosage...

Embodiment 3

[0056] (1) Preparation of DMEM medium: Dissolve DMEM dry powder in deionized water, stir until completely dissolved, adjust the pH value to 7.8, and sterilize and filter with a sterile filter membrane of ≤0.22 μm, and place it in a temperature of ≤4°C Refrigerator for standby use;

[0057] (2) adding the fetal bovine serum that accounts for 1-8% of the volume of the DMEM medium in the above-mentioned DMEM medium, and placing the DMEM medium in the culture vessel to carry out the cultivation of Vero monolayer cells;

[0058] (3) Discard the DMEM medium in the culture vessel where the monolayer cells are formed, and wash 2-3 times with a washing solution; the washing solution is sterile deionized water.

[0059] (4) Discard the washing solution, and add porcine trypsin, cell affinity agent and DMEM medium in step (1) to the culture vessel formed with monolayer cells, wherein the consumption of porcine trypsin is the volume consumption of DMEM medium 0.5% of DMEM, and the dosage...

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Abstract

The invention discloses a method for culturing a porcine epidemic diarrhea virus. The method comprises the following steps: (1) preparing a DMEM culture medium; (2) adding 1-8% of fetal bovine serum in the DMEM culture medium and carrying out monolayer cell culture; (3) discarding the DMEM culture medium inside a culture container formed with monolayer cells and washing with a washing liquid; (4) discarding the washing liquid and adding porcine trypsin, a cell affinity agent and the DMEM culture medium into the culture container formed with monolayer cells; and (5) placing the DMEM culture medium in the step (4) in a cell incubator, carrying out static culture for 1 minute-30 minutes, after the static culture is completed, taking the DMEM culture medium out, rapidly inoculating the porcine epidemic diarrhea virus, uniformly mixing, placing in the cell incubator, culturing and collecting the porcine epidemic diarrhea virus liquid. According to the method, by mainly changing the micro-relationship between the virus and micro cells during the culture, the affinity between the virus and the cells is increased so that the cells are more easily infected by the virus.

Description

technical field [0001] The invention relates to the field of microbial viruses, and more particularly, to a method for culturing porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea virus has seriously affected the pig industry in my country. It is highly infectious and harmful. It is one of the most important viruses affecting the global pig industry. The mortality rate of suckling piglets can reach 100%. Appears in the form of poison without symptoms. Porcine epidemic diarrhea virus belongs to group 1 of the genus Coronaviridae, transmissible gastroenteritis, feline coronavirus and canine coronavirus belong to the same genus of coronaviruses (Utiger et al., 1995a). Porcine Epidemic Diarrhea Virus was first discovered in the 1970s. The first discovered virus was isolated, purified and passaged and named as CV777. Many vaccine production units are still using this strain as a vaccine strain. In recent years, there has been a clinical outb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 冯晓声贾爱卿王贵平
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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