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Culture method for increasing titer of porcine epidemic diarrhea viruses

A technology of porcine epidemic diarrhea and culture methods, applied in the direction of microorganism-based methods, viruses, culture processes, etc., can solve the problems of low virus toxicity, achieve low virus toxicity, increase effective virus content, and protect invariance Inactivation effect

Active Publication Date: 2020-02-04
SICHUAN HUASHEN ANIMAL BIOLOGICAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a cultivation method for increasing the virulence of porcine epidemic diarrhea virus, which can significantly increase the virus content, avoid virus inactivation, and effectively solve the problem of low virulence of the virus

Method used

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  • Culture method for increasing titer of porcine epidemic diarrhea viruses

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Embodiment 1

[0019] A culture method for improving porcine epidemic diarrhea virus virulence, comprising the following steps:

[0020] (1) Vero cells were subcultured in a bottle at a volume ratio of 1:3, and then inserted into 1300 mL of DMEM medium containing 8vt% newborn bovine serum and a pH value of 7.2, and cultured at 36.5°C and 9r / h for 45h. A dense monolayer was obtained;

[0021] (2) pour out the used DMEM medium, then inoculate the porcine epidemic diarrhea virus of 1vt% in the dense monolayer of step (1) gained, cultivate 50min under 36.5 ℃ of temperature, 9r / h rotating speed condition, obtain mixture ;

[0022] (3) Add 1400mL of DMEM medium containing 1vt% newborn calf serum, 8 μg / mL insulin and 7g / L trehalose, and a pH value of 7.2 to the mixture obtained in step (2). Under cultivation, when the lesion rate is greater than 80%, the virus is harvested to obtain porcine epidemic diarrhea virus.

Embodiment 2

[0024] A culture method for improving porcine epidemic diarrhea virus virulence, comprising the following steps:

[0025] (1) The Vero cells were subcultured in a bottle at a volume ratio of 1:3, and then inserted into 1500 mL of DMEM medium containing 10vt% newborn bovine serum and a pH value of 7.3, and cultured for 48 hours at a temperature of 36.5°C and a rotational speed of 10r / h. A dense monolayer was obtained;

[0026] (2) pour out the used DMEM medium, then inoculate the porcine epidemic diarrhea virus of 2vt% in the dense monolayer of step (1) gained, cultivate 60min under 36.5 ℃ of temperature, 10r / h rotating speed condition, obtain mixture ;

[0027] (3) Add 1500mL of DMEM medium containing 2vt% newborn calf serum, 10μg / mL insulin and 8g / L trehalose, and a pH value of 7.3 to the mixture obtained in step (2), at a temperature of 36.5°C and a rotation speed of 10r / h Under cultivation, when the lesion rate is greater than 80%, the virus is harvested to obtain porcine...

Embodiment 3

[0029] A culture method for improving porcine epidemic diarrhea virus virulence, comprising the following steps:

[0030] (1) The Vero cells were passaged in bottles at a volume ratio of 1:4, and then inserted into 1600 mL of DMEM medium containing 12vt% newborn bovine serum and a pH value of 7.4, and cultured at 37.5°C and 11r / h for 50h. A dense monolayer was obtained;

[0031] (2) pour out the used DMEM medium, then inoculate the porcine epidemic diarrhea virus of 3vt% in the dense monolayer of step (1) gained, cultivate 70min under the condition of 37.5 ℃ of temperature, 11r / h rotating speed, obtain mixture ;

[0032] (3) Add 1600mL of DMEM medium containing 3vt% newborn bovine serum, 12μg / mL insulin and 9g / L trehalose, and a pH value of 7.4 to the mixture obtained in step (2), at a temperature of 37.5°C and a rotational speed of 11r / h Under cultivation, when the lesion rate is greater than 80%, the virus is harvested to obtain porcine epidemic diarrhea virus.

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Abstract

The invention discloses a culture method for increasing the titer of porcine epidemic diarrhea viruses. The culture method includes the following steps: Vero cells are subjected to bottle-separation passage, and then inoculated into cell growth fluid containing newborn calf serum to be cultured to obtain a dense single layer; then the porcine epidemic diarrhea viruses are inoculated into the densesingle layer to be cultured to obtain a mixture; and maintenance media containing the newborn calf serum, insulin and trehalose are added into the mixture, rotating culture is conducted, when the lesion rate is greater than 80%, the viruses are obtained, and thus the porcine epidemic diarrhea viruses are obtained. The content of the viruses can be significantly increased, virus inactivation is avoided, and the problem of low virus titer is effectively solved.

Description

technical field [0001] The invention relates to the technical field of porcine virus cultivation, in particular to a cultivation method for increasing the virulence of porcine epidemic diarrhea virus. Background technique [0002] Porcine Epidemic Diarrhea Virus (PEDV) belongs to class Ⅰ coronavirus, which causes porcine epidemic diarrhea disease. Piglets infected with this virus have a high mortality rate. The morphological characteristics of PEDV in intestinal epithelial cells are the same as those of other coronaviruses, and the virus is assembled by budding through the endoplasmic membrane. However, the virus particles detected in fecal samples are pleomorphic and tend to be spherical, with a diameter of about 95-190nm (including fibrils). Most virions have an electron-opaque central region with swollen fibrils 18-23 nm long, arranged radially outward from the nucleocapsid. When producing the virus liquid antigen of porcine epidemic diarrhea virus (PEDV), although it c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/071C12R1/93
CPCC12N5/0686C12N7/00C12N2500/34C12N2501/33C12N2770/20051
Inventor 肖昌建张传明邝声耀阴文奇周远成王子健
Owner SICHUAN HUASHEN ANIMAL BIOLOGICAL PRODS
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