Method for greatly increasing PCV2 cultivation virus titer through streptococcus culture preparation

A streptococcus and culture technology, applied in microorganism-based methods, biochemical equipment and methods, single-stranded DNA viruses, etc., can solve the problem of increasing production links, manpower input and waste discharge, increasing raw material input, and production costs. High cost and other problems, to achieve the effect of rapid improvement of toxin production capacity, increased possibility of pollution, and improvement of product quality

Active Publication Date: 2017-03-22
WUHAN ENG SCI & TECH RESINST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this procedure not only increases the input of raw materials, but also increases the production process, manpower input and waste discharge, which makes the production cost at least doubled. at 10 4 Below, the immune effect is poor
European and American countries adopt screening of highly sensitive cell lines to circovirus to cultivate circovirus and carry out virus concentration, the virus content of which is 10 6 Above, the immune effect is better, but its production cost is high, so its sales price is often 3-5 times that of domestic vaccines

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 can greatly improve the preparation of the Streptococcus culture preparation of PCV2 culture poison price

[0023] 1. Materials and methods

[0024] (1) Seed culture and main reagents:

[0025] PK-15 cells, Streptococcus agalactiae: purchased from CCTCC.

[0026] PBS powder: market purchase, domestic product.

[0027] Neonatal bovine serum, DMEM medium: purchased from Hyclone Company.

[0028] Trypsin: purchased from sigma company.

[0029] (2) Main operation steps and methods:

[0030] ①Preparation of adherent cells: PK-15 cells were thawed according to conventional adherent cell culture methods, cultured in Kjeldahl flasks, subcultured to the required number, and set aside.

[0031] ② Streptococcus inoculation: the above-mentioned adherent cells were subcultured for the last time and cultured for 48 hours, then the culture medium was changed to a maintenance medium containing less than 2% serum (DMEM medium containing less than 2% newborn bovine seru...

Embodiment 2

[0041] Example 2 Streptococcus and adherent cell co-culture preparation stimulates PCV2 culture system toxin production effect observation

[0042] 1. Materials and methods

[0043] (1) Seed culture and main reagents

[0044] PK-15 cells, PCV2 virus: all purchased from CCTCC.

[0045]PBS powder, D-glucosamine, PCV2 special PCR detection kit: market purchase, domestic product.

[0046] Neonatal bovine serum, DMEM medium: products of Hyclone Company.

[0047] Trypsin: product of sigma company.

[0048] (2) Main operation steps and methods

[0049] ①Host cell preparation: PK-15 cells were thawed according to conventional methods, cultured in Kelvin flasks, and passaged to the required number.

[0050] ② Virus inoculation: PCV2 was inoculated into the above PK-15 cells according to conventional methods, and cultured for 24 hours.

[0051] 3. Preparation of special culture medium for stimulating PCV2 toxin production: the streptococcus culture preparation prepared in Example ...

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Abstract

The invention discloses a method for greatly increasing the PCV2 cultivation virus titer through a streptococcus culture preparation, and belongs to the technical field of cell engineering and virus cultivation. The streptococcus culture preparation is prepared through high-temperature inactivation of streptococcus-containing supernate which is obtained after co-culture of streptococcus and anchorage-dependent cells in a maintenance culture medium. The streptococcus is streptococcus agalactiae, the cells are PK-15 cells, and the content of the streptococcus in the streptococcus-containing supernate is 100-900 million/mL. The streptococcus culture preparation is added into the maintenance culture medium according to the concentration of 0.1-1% to prepare a special culture medium used for stimulating PCV2 to generate viruses, the cultivation virus titer of the culture medium can be greatly increased by adopting the special culture medium as the maintenance culture medium for culturing PCV2, and the virus titer can reach 10<-7> or above; and a production process is greatly simplified, and the production cost is greatly lowered.

Description

technical field [0001] The invention belongs to the technical field of cell engineering and virus culture, in particular, the invention relates to a streptococcus culture preparation capable of greatly increasing the culture toxicity of PCV2 (porcine circovirus type 2) and its preparation method and the use of the preparation to improve Method for culturing virulence of PCV2. Background technique [0002] At present, there are mainly two types of porcine circovirus vaccines in use in China—subunit vaccines and whole virus vaccines. Although subunit vaccines are relatively safe, they are expensive to produce and have a short immunity period. Therefore, the vast majority of pig farms in China still rely mainly on whole virus vaccines. However, the production of whole-virus vaccines also has its technical bottlenecks: circovirus cultured cells in vitro have low infectivity and slow infection speed, and the replication speed of the virus in different individual cells varies gre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N5/071C12N7/00C12R1/93C12R1/465
CPCC12N1/20C12N5/0686C12N7/00C12N2502/70C12N2750/10051
Inventor 高其双彭霞谭珺隽夏瑜程百炼桑奥文雷长宁杨俊红王利燕田晨雪
Owner WUHAN ENG SCI & TECH RESINST
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