Preparation method of TGEV

A virus and microcarrier technology, applied in the field of cell engineering, can solve problems such as low titer of TGEV virus, and achieve the effects of uncomplicated cell harvesting process, low production cost and high cell yield

Inactive Publication Date: 2016-03-16
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the technical defect of the prior art, provides a kind of TGEV virus preparation method, to solve the technical problem that the TGEV virus titer prepared by the related preparation method of the prior art is low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Bioreactor: 14L bioreactor from Berenger, Germany.

[0032] Microcarrier: Cytodex-1 (purchased from GE).

[0033] Porcine transmissible gastroenteritis virus: TGEVHB08 strain.

[0034] Cell growth medium: MEM containing 8% newborn bovine serum by volume;

[0035] Virus maintenance solution: MEM containing 2% newborn bovine serum by volume;

[0036] Microcarrier suspension culture of ST cells: In a 14L bioreactor, add the treated microcarrier Cytodex-1 at a concentration of 2g / L, and inoculate ST cells with a cell inoculation concentration of 400,000 / mL. The optimal setting parameters of the reactor during cell culture: pH7.25, temperature 37°C, dissolved oxygen 55%, stirring speed 35rpm / min. After ST cells were inoculated, samples were taken at regular intervals, the state of the cells was observed under a microscope, and the cells were counted to determine the consumption of glucose. According to the situation of glucose consumption and cell growth and detachment, ...

Embodiment 2

[0040] Bioreactor: 14L bioreactor from Berenger, Germany.

[0041] Microcarrier: Cytodex-1 (purchased from GE).

[0042] Porcine transmissible gastroenteritis virus: TGEVHB08 strain.

[0043] Cell growth medium: MEM containing 10% newborn calf serum by volume;

[0044] Virus maintenance solution: MEM containing 3% newborn bovine serum by volume;

[0045] Microcarrier suspension culture of ST cells: In a 14L bioreactor, add the treated microcarrier Cytodex-1 at a concentration of 8g / L, and inoculate ST cells with a cell inoculation concentration of 1 million / mL. The optimal setting parameters of the reactor during cell culture: pH7.0, temperature 37°C, dissolved oxygen 55%, stirring speed 80rpm / min. After ST cells were inoculated, samples were taken at regular intervals, the state of the cells was observed under a microscope, and the cells were counted to determine the consumption of glucose. According to the glucose consumption and the growth and shedding of cells, the mediu...

Embodiment 3

[0049] A kind of TGEV virus preparation method, comprises the following steps:

[0050] 1) Get the microcarrier and place it in PBS buffer solution for soaking treatment, then sterilize, discard the PBS buffer solution, add it to the MEM cell growth solution containing 9% (w / w) serum, and obtain the mixed culture medium for subsequent use. The density of the microcarrier in the medium is 4g / L;

[0051] 2) Inoculate 0.6×10 6 Cells / mL ST cells, and then cultured at pH 6.6, temperature 35°C, dissolved oxygen 40%, stirring speed 35rpm;

[0052] 3) When the cell density reaches 5×10 6 Cells / mL, discard the cell growth solution in the mixed medium, add the virus maintenance solution, and then inoculate it with 1.5% porcine transmissible gastroenteritis virus, at a temperature of 36°C, dissolved oxygen 40%, and a stirring speed of 35rpm Under culture, wherein the virus maintenance solution is MEM cell growth solution containing 1% serum;

[0053] 4) When the virus is inoculated a...

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Abstract

The invention provides a preparation method of TGEV (transmissible gastroenteritis virus). According to the method, in a bioreactor, an ST cell is adopted as the host cell, microcarrier culture is employed for proliferation of TGEV, and at the same time efficient condition parameters are designed. The culture efficiency is significantly improved, the cell density is enhanced by 3-5 times than spinner bottles, the virus titer is high, and is increased from the 10<7.0-7.3>TCID50/ml of spinner bottle technique to 10<7.7>-10<8.5>TCID50/ml, and the titer is enhanced by 5-10 times. At the same time, a high titer antigen needs a high dilution factor during use, so that the content of heterologous protein in a unit volume antigen is reduced indirectly, thereby lowering the side reaction incidence of vaccines prepared with heterologous protein as the antigen. The technology is low in cost, and compared with the practice of selecting a traditional basic medium, the harvest time is greatly shortened, the interassay difference is small, and the process is stable.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a preparation method of TGEV virus. Background technique [0002] Porcine transmissible gastroenteritis (TGE) is a highly contagious enteric disease caused by porcine transmissible gastroenteritis virus (TGEV), with vomiting, severe diarrhea and dehydration as the main clinical features. Pigs of different ages and breeds are susceptible to infection, and the mortality rate of piglets within 2 weeks of age can reach 100%, which is the main infectious disease that causes piglets to become ill and die. The disease mainly occurs in winter and early spring, and is widely prevalent in many countries and regions in the world. It can be mixed with porcine rotavirus disease and porcine epidemic diarrhea. In recent years, the epidemic trend has gradually increased, and TGEV is still one of the main causes of piglet morbidity and death, causing great troubles to the pig industry, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/225A61P31/14C12R1/93
CPCC12N7/00A61K39/12C12N2770/20021C12N2770/20034
Inventor 宋磊吕茂杰杨保收付旭彬梁武
Owner TIANJIN RINGPU BIO TECH
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