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Vero-E6 suspension cell strain sVero-E6 adapted to porcine epidemic diarrhea virus and application of Vero-E6 suspension cell strain sVero-E6

A technology for porcine epidemic diarrhea and suspension cells, which is applied in animal cells, antiviral agents, viruses/phages, etc., can solve the problems of time-consuming and labor-intensive, cumbersome operation, complicated process, etc. The effect of culture efficiency and high cell viability

Active Publication Date: 2022-04-05
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional adherent culture requires the use of cell spinner bottles and cell factories in the process of virus expansion and reproduction, which consumes a lot of manpower and financial resources
In the existing suspension culture method, for example, patent document CN107267443A (hereinafter referred to as document 1) discloses a Vero E6 cell strain adapted to full suspension culture, but it needs to replace the cell culture medium by centrifugation before the inoculation of porcine epidemic diarrhea virus It is a virus culture and maintenance medium containing 1-2% newborn bovine serum, which is cumbersome, time-consuming and labor-intensive, and a certain amount of trypsin needs to be added during PEDV culture, and the virus maintenance solution containing serum will affect the action of trypsin
Patent document CN108866012A (hereinafter referred to as document 2) discloses a production method for porcine epidemic diarrhea virus suspension culture and continuous poison collection, but it needs to use sheet carrier suspension culture, the cost is high, and the cultured Vero cells need to be washed before inoculation , it is necessary to change the liquid during inoculation, the process is complex, time-consuming and labor-intensive

Method used

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  • Vero-E6 suspension cell strain sVero-E6 adapted to porcine epidemic diarrhea virus and application of Vero-E6 suspension cell strain sVero-E6
  • Vero-E6 suspension cell strain sVero-E6 adapted to porcine epidemic diarrhea virus and application of Vero-E6 suspension cell strain sVero-E6
  • Vero-E6 suspension cell strain sVero-E6 adapted to porcine epidemic diarrhea virus and application of Vero-E6 suspension cell strain sVero-E6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: Domestication and identification of sVero-E6 suspension strain

[0046] 1.1. The Vero-E6 cells (provided by Jinyu Baoling Bio-Pharmaceutical Co., Ltd.) recovered and frozen were cultured for 2 generations with DMEM containing 10% newborn calf serum (NBCS), and the serum was reduced to 7%; Reduce to 4%; re-cultivate the serum for 3 generations to reduce to 3%, add 5% to 10% conditioned medium (DMEM containing 3% NBCS) in the culture solution after continuous culture for 3 generations, the ratio of subculture and bottle division is It is advisable to grow into a dense monolayer within 48 hours.

[0047] 1.2. In Step 1.1, the cells adapted to the 3% NBCS low-serum culture conditions were gradually replaced with serum-free DMEM medium to reduce the proportion of bottle division. Collect the cells suspended in the culture medium and the cells with poor adhesion. You can also collect the remaining cells after mild digestion with trypsin, and adjust the cell d...

Embodiment 2

[0059] Example 2: Proliferation and Culture of PEDV Using sVero-E6 Suspension Cells

[0060] 2.1. Proliferation and cultivation of PEDV in shake flasks

[0061] 2.1.1: Add the well-grown sVero-E6 suspension cells into the shake flask containing the same CD ST 258 nutrient solution as in 1.5.1, at 35.0°C-37.0°C, 5.0% CO 2 , 100rpm / min-130rpm / min shaker culture for 48h-72h, carry out subculture and enlargement culture;

[0062] 2.1.2: When the cell density reaches (4.0-6.0)×10 in step 2.1.1 6 Cells / mL, when the viability rate reaches above 95.0%, add virus maintenance solution with the same volume as the nutrient solution CD ST 239 (serum-free virus maintenance solution, purchased from Gansu Jianshun Biotechnology Co., Ltd.), the cell density was (2.0-3.0) × 10 6 cells / mL;

[0063] 2.1.3: Add EDTA-trypsin with a final concentration of 5 μg / ml-25 μg / ml and 1.0%-5.0% PEDV seed poison ZJ-08 (preserved in Jinyu Baoling Biology) to the culture medium in step 2.1.2 Pharmaceuti...

Embodiment 3

[0112] Example 3: Comparison of the virulence of PEDV inoculated in Vero-E6 adherent cells and sVero-E6 suspension cells

[0113] 3.1. Culture PEDV with Vero-E6 adherent cells

[0114] 3.1.1: Digest and disperse Vero-E6 adherent cells with trypsin, add cell culture medium (DMEM+10% newborn bovine serum), prepare a cell suspension with a ratio of 1:3 for passage, and pack in cell bottles , 37°C, 5% CO 2 Cultivate in an incubator for 3-4 days, and pass passage after forming a dense monolayer of cells.

[0115] 3.1.2: Preparation for inoculating cells: Digest and disperse the above-mentioned Vero-E6 cells forming a dense cell monolayer with trypsin, add cell culture medium, prepare a cell suspension with a ratio of 1:3, and pack in cell bottles Medium, 37°C, 5% CO 2 The incubator is cultivated for 48-72 hours, and the cells are required to cover a monolayer, grow well, and be healthy and pollution-free.

[0116] 3.1.3: Inoculation and adsorption: Discard the cell culture me...

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Abstract

The invention discloses a Vero-E6 suspension cell strain sVero-E6 adapted to a porcine epidemic diarrhea virus and application of the Vero-E6 suspension cell strain sVero-E6, and belongs to the technical field of biology. The sVero-E6 suspension strain provided by the invention can stably subculture and grow, when the sVero-E6 suspension strain is used for culturing PEDV, the cultured PEDV can stably proliferate, and the sVero-E6 suspension strain can be used for continuously proliferating and culturing the PEDV and repeatedly harvesting a virus culture solution, so that a large amount of production cost and culture time can be saved, and the productivity is improved. The inactivated vaccine prepared from the obtained virus culture solution is good in immunogenicity, stable and safe, and can be effectively used for preventing porcine epidemic diarrhea diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Vero-E6 suspension cell strain (named sVero-E6 suspension strain) adapted to porcine epidemic diarrhea virus and its application, in particular to the cultivation of porcine epidemic diarrhea virus and the production of porcine epidemic diarrhea virus. Application of inactivated diarrhea virus vaccine. Background technique [0002] Porcine Epidemic Diarrhea (PED) is a highly contagious intestinal infectious disease caused by Porcine Epidemic Diarrhea Virus (PEDV), and pigs of all ages are susceptible to PEDV. Serious economic losses are caused to the pig industry. [0003] At present, porcine epidemic diarrhea disease is usually prevented by vaccination. In China, Vero-E6 cells (African green monkey kidney cells) are often used for adherent or suspension culture and propagation of porcine epidemic diarrhea virus to produce related vaccines for preventing porcine epidem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12N7/04A61K39/215A61P31/14
CPCY02A50/30
Inventor 李静刘建奇俎红利宋庆庆
Owner JINYUBAOLING BIO PHARMA CO LTD
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