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Preparation method of porcine epizootic diarrhea virus

A porcine epidemic diarrhea and virus technology, applied in the field of porcine epidemic diarrhea virus preparation, can solve the problems of affecting the culture effect and unsatisfactory host cell performance, and achieve high culture density, high production virus titer, and high degree of automation control Effect

Inactive Publication Date: 2017-12-08
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a method for preparing porcine epidemic diarrhea virus, so as to solve the technical problem that the culture effect is affected by the unsatisfactory host cell performance in the PEDV culture method of the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Bioreactor: 14L bioreactor from Berenger, Germany.

[0037] Microcarrier: Cytodex-1 (purchased from GE).

[0038] Porcine epidemic diarrhea virus: PEDVZJ08 strain, CV777 strain or not limited to this strain.

[0039] Cell growth medium: DMEM containing 10% newborn bovine serum by volume;

[0040] Cell maintenance solution: DMEM containing 2% newborn calf serum by volume;

[0041] Cell selection: select IPEC-J2 cells as cells for multiplying viruses;

[0042] Cell subculture and culture: the above cells were digested and subcultured with EDTA-trypsin cell dispersion solution (PBS solution containing 0.25% trypsin (specification 1:250), 0.02% EDTA), and replaced with 90% DMEM solution and 10% calf serum , the cell growth solution with a pH of 7.2 continued to culture, and the culture temperature was 37°C. When a good monolayer is formed, it can be used for continuous subculture or inoculated in a bioreactor for microcarrier suspension culture.

[0043] Proliferation of...

Embodiment 2

[0048] A preparation method for porcine epidemic diarrhea virus, comprising the following steps:

[0049] 1) Take IPEC-J2 cells, use EDTA-trypsin dispersion solution to digest and disperse, then add cell growth medium and culture at 36°C;

[0050] 2) When the IPEC-J2 cells have grown to a monolayer, insert 0.1% PEDV into it, and continue to cultivate until the IPEC-J2 cell lesion rate exceeds 50%, and the virus titer in the culture exceeds 10%. 7.5 TCID 50 / mL, the seed poison for production is obtained;

[0051] 3) In the stirred bioreactor, the microcarrier was mixed with the cell growth solution in an amount of 2g / L to obtain a mixed culture medium, and then 0.3×10 6 Each / mL IPEC-J2 cells were cultured under the conditions of pH 6.5, temperature 36°C, dissolved oxygen 40%, stirring speed 30rpm;

[0052] 4) When the cell density in the mixed medium reaches 2×10 6 When cells / mL, discard the cell growth solution in the mixed culture medium, add cell maintenance solution, i...

Embodiment 3

[0063] A kind of porcine epidemic diarrhea virus preparation method is characterized in that comprising the following steps:

[0064] 1) Take IPEC-J2 cells, use EDTA-trypsin dispersion solution to digest and disperse, then add cell growth medium and culture at 38°C;

[0065] 2) When the IPEC-J2 cells have grown to a monolayer, insert 5% PEDV therein, and continue to cultivate until the IPEC-J2 cell lesion rate exceeds 50%, and the virus titer in the culture exceeds 10%. 7.5 TCID 50 / mL, the seed poison for production is obtained;

[0066] 3) In the stirred bioreactor, the microcarrier was mixed with the cell growth liquid in an amount of 10 g / L to obtain a mixed culture medium, and then 1 × 10 6 Cells / mL of IPEC-J2 cells were cultured under conditions of pH 7.5, temperature 38°C, dissolved oxygen 80%, and stirring speed 90 rpm;

[0067] 4) When the cell density in the mixed medium reaches 5×10 6 When cells / mL, discard the cell growth liquid in the mixed culture medium, add...

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Abstract

The invention provides a preparation method of a porcine epizootic diarrhea virus (PEDV). According to the technical scheme, the ideal host cell of the PEDV is inspected based on cell growth rate and culture product virus valence, and the scheme for performing PEDV cultivation by IPEC-J2 cells instead of Vero cells is finally determined. Experiments found that the IPEC-J2 cells and the PEDV have higher sensitivity, the lesion rate of the cells after the viruses are inoculated is higher, meanwhile, the viruses have higher replication rate in the cells, and the growth rate of the IPEC-J2 cells is higher, so the process flow can be shortened. In the cultivation process, various defects in the traditional spinner cultivation are analyzed, large-scale cultivation is conducted by creatively adopting a stirring type bioreactor, meanwhile, for the biological characteristics of the IPEC-J2 cells and the PEDV, proper conditions are matched from the angles of microcarrier adding amount, cell inoculation density, virus inoculation quantity, cell density during virus inoculation, virus receiving time, reactor operating parameters and the like, so that the virus cultivation efficiency is obviously improved.

Description

technical field [0001] The invention relates to the technical field of veterinary biological products, further relates to the cultivation and propagation of viruses, and in particular to a method for preparing porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea (PED) is a highly contagious disease caused by porcine epidemic diarrhea virus (PEDV). Its clinical characteristics are vomiting, diarrhea and dehydration in piglets. The pathological changes are mainly manifested in the jejunum and ileum of pigs. The intestinal villi atrophy and fall off, pigs of all ages can be affected, the most serious harm to suckling piglets, bring significant economic losses to the pig industry. [0003] In the prior art, the cultivation of porcine epidemic diarrhea virus is mainly based on Vero E6 cells as the host, and the lesion rate of the cells after inoculation with PEDV is low, and even if lesions occur, it is difficult to obtain a higher virus titer....

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2770/24351
Inventor 张桂红王和平樊翠付旭彬郁宏伟梁武李守军
Owner TIANJIN RINGPU BIO TECH
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