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Method for large scale cultivation in bioreactor

A large-scale culture and bioreactor technology, which can be quickly adapted to the field of MDCK cell culture within 5 generations of recombinant H5N1 avian influenza virus, can solve problems such as the inability of cultured virus titers to meet vaccine production, and achieve easy industrialized large-scale operation, cell Rapid proliferation and good virus immunogenicity

Active Publication Date: 2012-10-17
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for large-scale cultivation of a bioreactor, which is easy to implement and easy to operate, and the titer of virus HA after adaptation is >2 7 ; The virus is quickly converted from the square bottle and rotary bottle culture process to the large-scale culture of the simple-to-operate fixed-bed basket stirring system reactor, and its HA titer can reach 2 11 , to solve the problem that the virus titer of recombinant H5N1 avian influenza virus cultured on MDCK cells cannot meet the requirements of vaccine production

Method used

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  • Method for large scale cultivation in bioreactor
  • Method for large scale cultivation in bioreactor
  • Method for large scale cultivation in bioreactor

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Recombinant H5N1 avian influenza virus strain rC4 / W1 strain rapidly adapted to MDCK cells

[0025] rC4 / W1 strain (see Genetic Resources Table): This virus is a fully avian-derived H5 subtype avian influenza virus. It is preserved in the China Center for Type Culture Collection with the preservation number: CCTCC NO: V201029. The steps for its rapid adaptation to MDCK cells are as follows :

[0026] 1) One MDCK cell was grown into a dense monolayer T75 cell flask, and the cells were digested and evenly spread into three 6-well cell culture plates.

[0027]2) Chicken embryo allantoic fluid venom is diluted to 10 times with 10 times ratio -6 .

[0028] 3) Take 10 -3 、10 -4 、10 -5 、10 -6 Each dilution of venom was 200ul, supplemented to 3ml with cell maintenance solution, and added to a 6-well plate full of MDCK cell monolayers, each dilution concentration was repeated, and 200ul of the dilution was supplemented to 3ml with cell maintenance solution, and ad...

Embodiment 2

[0035] Example 2: Rapid Adaptation of Recombinant H5N1 Avian Influenza Virus Strain XN / PR8 (See Genetic Resources Table) to MDCK Cells

[0036] 1) One MDCK cell was grown into a dense monolayer T75 cell flask, and the cells were digested and evenly spread into three 6-well cell culture plates.

[0037] 2) Chicken embryo allantoic fluid venom is diluted to 10 times with 10 times ratio -8 .

[0038] 3) Take 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Each dilution of venom was 200ul, supplemented to 3ml with cell maintenance solution, and added to a 6-well plate full of MDCK cell monolayers, each dilution concentration was repeated, and 200ul of the dilution was supplemented to 3ml with cell maintenance solution, and added to a long A monolayer full of MDCK cells was used as a negative control.

[0039] 4) At 37°C, remove the virus solution after virus adsorption for 1.5 hours, wash the MDCK cells with virus dilution solution for 3 times, add 3ml of cell maintenance solution to eac...

Embodiment 3

[0045] Example 3: Recombinant H5N1 avian influenza virus strain XN / W1 rapidly adapts to MDCK cells

[0046] The XN / W1 strain (see Genetic Resources Table) belongs to the whole bird-derived H5 subtype avian influenza virus. The steps of its rapid adaptation to MDCK cells are as follows:

[0047] 1) One MDCK cell was grown into a dense monolayer T75 cell flask, and the cells were digested and evenly spread into three 6-well cell culture plates.

[0048] 2) Chicken embryo allantoic fluid venom is diluted to 10 times with 10 times ratio -6 .

[0049] 3) Take 10 -3 、10 -4 、10 -5 、10 -6 Each dilution of venom was 200ul, supplemented to 3ml with cell maintenance solution, and added to a 6-well plate full of MDCK cell monolayers, each dilution concentration was repeated, and 200ul of the dilution was supplemented to 3ml with cell maintenance solution, and added to a long A monolayer full of MDCK cells was used as a negative control.

[0050] 4) At 37°C, remove the virus solutio...

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Abstract

The invention discloses a method for large scale cultivation in a bioreactor, comprising the following steps: (1) making a recombinant H5N1 avian influenza virus adapt to MDCK cells; (2) preparing a seed lot by using the adapted virus strain as a reactor large-scale culture virus, wherein the seed lot virus generation is no less than F6, the hemagglutination titer is no less than 28, and EID50 is no less than 107.5; (3) culturing the MDCK cells by using polyester sheets as a carrier and the reactor as a basket fixed bed, and pouring the cells on every gram of the carrier, wherein the culture medium is newborn calf serum DMEM; (4) inoculating the virus on the cultured MDCK cells, washing the cells by using a virus diluent, conducting virus inoculation, after absorption, and replacing a cell maintenance media; (5) maintaining the MDCK cells and conducting virus propagation, stirring, and replacing the cell maintenance media; and (6) monitoring the hemagglutination titer and harvesting the virus, and harvesting the reactor to culture a volume virus liquid. According to the invention, the method is easy to operate and the operation is simple, the HA titer of the virus after adaption is higher than 2<7>, and the problem that the culture virus titer cannot satisfy the demand of produce vaccine in the existing culture of the recombinant H5 subtype avian influenza virus on the MDCK cells is solved.

Description

technical field [0001] The invention belongs to the field of veterinary biotechnology, and more specifically relates to a method for rapidly adapting to MDCK cell culture within 5 generations of recombinant H5N1 avian influenza virus, which is suitable for large-scale culture of virus strains in bioreactors. technical background [0002] H5N1 highly pathogenic avian influenza virus (HP-AIV) exists widely in the world, not only can infect poultry, but also can infect a variety of mammals across the interspecies barrier, especially can directly infect humans, And cause death, seriously endanger the poultry industry and human health, so the prevention and control of bird flu is very important. Mass culling and biosafety protection are the main measures for timely extermination and control of highly pathogenic avian influenza (HPAI), while vaccination is the most effective and economical method for preventing and controlling avian influenza. [0003] Influenza vaccines have bee...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/93
Inventor 金梅林李国红蒋桃珍徐高原李俊平周明光洪灯左静涂加刚邸海波韦大坤徐玲莉陈焕春
Owner WUHAN KEQIAN BIOLOGY CO LTD
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