BHK-21 cell line BHK-21-BP-2 clones and application thereof in full suspension cell culture

A technology of BHK-21-BP-2 and BHK-21, applied in the field of veterinary biological products, can solve the problems of unsuitability for large-scale production of vaccines, large differences between vaccine batches, and occupying a lot of space, and achieve excellent immunogenicity and security, high integrity, high integrity effect

Active Publication Date: 2019-08-23
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of domestic production of pseudorabies vaccine mainly adopts the spinner bottle culture process, but due to the shortcomings of large batch-to-batch differences, low production efficiency, and large space occupation, this method is increasingly unsuitable for the current large-scale production of vaccines.

Method used

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  • BHK-21 cell line BHK-21-BP-2 clones and application thereof in full suspension cell culture
  • BHK-21 cell line BHK-21-BP-2 clones and application thereof in full suspension cell culture
  • BHK-21 cell line BHK-21-BP-2 clones and application thereof in full suspension cell culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Suspension acclimatization of BHK-21 adherent cells

[0048] The 3 strains of BHK-21 adherent cell clones from different sources that were cryopreserved were resuscitated, and the DMEM culture medium containing 8% (v / v) fetal calf serum (product of Hyclone Company, product code: SH30022.01) was used in a 75cm 2 Cell culture flasks. When the cells grow into a dense monolayer and the growth state is good, discard the original culture medium, add 0.25% trypsin for digestion, blow and disperse the cells into a single cell suspension with a pipette gun, and dilute the cells with 1×10 6 The density of cells / ml was respectively inserted into 250ml Erlenmeyer shake flasks, and the culture medium was cultured by using the original culture medium (DMEM containing 8% (v / v) fetal calf serum) and the suspension medium BHK201 mixed in different proportions. During the cultivation process, gradually reduce the ratio of the original medium to the suspension medium, and the r...

Embodiment 2

[0053] Example 2 Screening and performance analysis of 3 strains of BHK-21 suspension clones

[0054] 1. Toxicity analysis

[0055] BHK-21-BP-1, BHK-21-BP-2, and BHK-21-BP-3 obtained in Example 1 were cultured in 250ml cell shake flasks respectively, and the suspension culture The culture medium is BHK201, the culture volume is 30-50ml, and the culture condition is 5% CO 2 , 37°C, 120rpm. After culturing for 48h, the ZY-2014 strain of pseudorabies virus was inoculated respectively according to MOI=0.5, and the cells in different growth stages were sampled, counted and determined for 0h before inoculation, 24h, 40h, 48h, 72h, and 96h after inoculation ( supernatant). When sampling, there is no need to freeze and thaw, and the supernatant poison value of the sample is directly measured after the sample is centrifuged.

[0056] The result is as figure 1 and figure 2 As shown, compared with the other two clones, the suspension cell BHK-21-BP-2 clone had a lower number of cu...

Embodiment 3

[0085] Example 3 BHK-21 cell line BHK-21-BP-2 clone suspension cells cultured in a bioreactor

[0086] The BHK-21 cell line BHK-21-BP-2 clone was cultured in a shake flask for 1 to 3 generations, expanded to the number of cells required for inoculation in a 10L bioreactor, transferred to a 10L bioreactor, and Add suspension medium BHK201 to make the final cell concentration 0.4~0.8×10 6 cells / ml. Under the conditions of 36.5±0.5℃, pH value 7.2±0.1, DO value 30%~60%, stirring speed 60rpm~150rpm (stirring speed varies with cell density), culture for 48h~72h, when the cells in the reactor The density reaches 3~6×10 6 cells / ml, it is used for further amplified culture or inoculation.

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Abstract

The invention belongs to the technical field of veterinary biological products, and particularly relates to a BHK-21 cell line BHK-21-BP-2 clone and application thereof in full suspension cell culture. The provided BHK-21-BP-2 clone is preserved in the China general microbiological culture collection center, and the preservation number is CGMCC No. 17588. After the clone is inoculated with pseudorabies viruses during full suspension cell culture, the clone has rapid cytopathy, the virulence values of generated viruses are high, and the viruses can be collected after 24-48 hours; at the time ofvirus collection, only supernatant needs to be collected by centrifugation without repeated freezing and thawing, and the production process is simple. The total protein content of a produced virus antigen solution is low, the integrality and quality of obtained effective viral antigens are high, the viruses are excellent in immunogenicity and safety, and a prepared pseudorabies virus vaccine hasthe advantages of high stability, small difference between batches, a good immune effect and high safety.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a BHK-21 cell line BHK-21-BP-2 clone and its application in full suspension cell culture. Background technique [0002] Pseudorabies is an acute infectious disease of various domestic and wild animals caused by Pseudorabies virus (PRV). Pigs are the main host of the virus. Newborn piglets and piglets under 15 days of age show obvious neurological symptoms, and the mortality rate reaches 100%. Fattening pigs often show invisible infection, and adult pigs are more resistant to infection after being infected, but long-term detoxification. For miscarriage, stillbirth, mummified fetus, etc. In recent years, due to the prevalence of new mutant strains of pseudorabies virus, pseudorabies has occurred in many pseudorabies vaccine immunized pig farms. The method of domestic production of pseudorabies vaccine mainly adopts the spinner bottle culture pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/93
CPCC12N5/0686C12N7/00C12N2710/16711
Inventor 潘文黄书林杨君敬潘春刚申咏红
Owner CHINA ANIMAL HUSBANDRY IND
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