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A kind of cultivation method of 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: 2017-06-30
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A kind of cultivation method of porcine epidemic diarrhea virus
  • A kind of cultivation method of porcine epidemic diarrhea virus
  • A kind of cultivation method of porcine epidemic diarrhea virus

Examples

Experimental program
Comparison scheme
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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 use a sterile filter membrane of ≤0.22 μm for sterile filtration, and place it in a room of ≤4°C Refrigerator ready for use;

[0045] (2) adding fetal calf serum accounting for 1-8% of the volume of the DMEM medium in the above-mentioned prepared DMEM medium, and placing the DMEM medium in a culture vessel for culturing Vero monolayer cells;

[0046] (3) Discard the DMEM medium in the culture container with the monolayer cells formed, and wash with washing liquid for 2-3 times; the washing liquid is sterile deionized water.

[0047] (4) Discard washing liquid, add porcine trypsin, cell affinity agent and the DMEM substratum in step (1) in the culture vessel that forms monolayer cell, wherein the consumption of porcine trypsin is DMEM substratum volume consumption 0.3% of DMEM medium volume, and the amount of cell affinity agent is ...

Embodiment 2

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

[0051] (2) adding fetal calf serum accounting for 1-8% of the volume of the DMEM medium in the above-mentioned prepared DMEM medium, and placing the DMEM medium in a culture vessel for culturing Vero monolayer cells;

[0052] (3) Discard the DMEM medium in the culture container with the monolayer cells formed, and wash with washing liquid for 2-3 times; the washing liquid is sterile deionized water.

[0053] (4) Discard washing liquid, add porcine trypsin, cell affinity agent and the DMEM substratum in step (1) in the culture vessel that forms monolayer cell, wherein the consumption of porcine trypsin is DMEM substratum volume consumption 0.1% of DMEM medium volume, and the amount of cell affin...

Embodiment 3

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

[0057] (2) adding fetal calf serum accounting for 1-8% of the volume of the DMEM medium in the above-mentioned prepared DMEM medium, and placing the DMEM medium in a culture vessel for culturing Vero monolayer cells;

[0058] (3) Discard the DMEM medium in the culture container with the monolayer cells formed, and wash with washing liquid for 2-3 times; the washing liquid is sterile deionized water.

[0059] (4) Discard washing liquid, add porcine trypsin, cell affinity agent and the DMEM substratum in step (1) in the culture vessel that forms monolayer cell, wherein the consumption of porcine trypsin is DMEM substratum volume consumption 0.5% of DMEM medium volume, and the amount of cell affinity a...

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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, in particular to a method for cultivating 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 after infection can reach 100%. Appears in the form of poison without showing symptoms. Porcine epidemic diarrhea virus belongs to group 1 of the genus Coronaviridae in the family Coronaviridae, and 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 named CV777 after isolation, purification and passage. Many vaccine production units are still using this strain as a vaccine str...

Claims

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Application Information

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Patent Type & Authority Patents(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