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Culturing method of porcine circovirus type 2-containing cells

A technology of porcine circovirus and a culturing method, which is applied in the field of culturing cells containing porcine circovirus type 2, can solve the problems of complex process, long development cycle, difficult product quality control, etc., and achieves a simple process and stable biological characteristics. Effect

Inactive Publication Date: 2014-12-24
上海佳牧生物制品有限公司 +2
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

At present, the main technical route of the research on the toxicity of the virus is to use D-glucosamine to treat the cells to enhance the proliferation ability of the virus, or to use the virus to continuously culture and pass on the cells, so that the isolates will increase with the number of cell passages, and the virus titer will increase. High; but the development cycle is long, the process is complicated, the product quality is difficult to control, and the cost is high, which is not conducive to large-scale production of vaccines with reasonable prices
At present, there is no report at home and abroad on the cultivation of porcine circovirus type 2 by limiting dilution and cell cloning of PK15 cells containing porcine circovirus type 2

Method used

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Embodiment

[0021] A culture method containing porcine circovirus type 2 cells, comprising the following steps:

[0022] (1) Culture of a single PK15 cell clone containing porcine circovirus type 2 and its subpopulations

[0023] The PCV2 / SN07-12 virus was inoculated into porcine kidney cells (PK15 cells) without type 1 porcine circovirus contamination and cultured in a 37°C incubator, and the monolayer containing pigs in the logarithmic growth phase was digested with trypsin. The PK15 cells of circovirus type 2 were then used to limit the dilution method. According to the cell growth medium containing 20wt% bovine serum in each well of 100uL, the density of 0.5 cells / well was added to a 96-well cell culture plate and placed in a 96-well cell culture plate containing 5vt% CO 2 , cultured in an incubator at a temperature of 37°C, select the cells in the cell wells with single cell growth for subcloning culture, digest with trypsin, add bovine serum cell growth medium, and then add 96-well ...

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Abstract

The invention relates to a culturing method of porcine circovirus type 2-containing cells. The method comprises the following steps: cloning of an individual porcine circovirus type 2-containing PK15 cell and culturing of a subgroup of the PK15 cell: inoculating PCV2 / SN07-12 seed virus into porcine circovirus type 1 pollution-free porcine kidney cells (PK15 cells), screening positive porcine circovirus type 2-containing PK15 cell cloning strains, and multiplying high-titer porcine circovirus type 2 to achieve virus titer up to 10<7.0>TCID50 / ml, wherein the microbial collection registration number of the PCV2 / SN07-12 seed virus is CCTCC NO:V201304. Compared with the prior art, the cells cultured by adopting the method have the advantages of being simple in technology, high in virus titer, stable in biological characteristics, and the like.

Description

technical field [0001] The invention belongs to a method for cultivating virus cells, in particular to a method for cultivating cells containing porcine circovirus type 2. Background technique [0002] The diseases caused by porcine circovirus type 2 (Porcine Circovirus type 2, PCV2) as the main pathogenic factor are collectively referred to as porcine circovirus disease (PCV2-associated disease, PCVAD). At present, PCVAD has occurred and spread all over the world, and it is one of the serious infectious diseases that endanger pig production. There is no effective preventive measure for the related diseases caused by PCV2, and the vaccine made with PCV2 antigen has achieved initial success in the prevention of porcine circovirus disease. With the formation and development of related vaccines, diagnostic reagents and treatment methods against PCV2, effective and reliable methods are needed to obtain sufficient PCV2 viruses. At present, the culture of PCV2 is established on ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00
Inventor 何锡忠李春华赵本进张春玲倪建平蒋凤英彭丽英张婉华
Owner 上海佳牧生物制品有限公司
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