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Method for proliferating avian influenza virus by adopting MDCK cell line

An avian influenza virus and cell line technology, applied in the field of cell culture, can solve the problems of inability to meet the needs of virus vaccine production, unsuitable for large-scale culture, complicated and tedious procedures, etc., to maintain sensitivity and biological characteristics, and strong virus proliferation ability. , the effect of high agglutination titer

Pending Publication Date: 2020-09-15
ZHAOQING DAHUANONG BIOLOGIC PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are some defects in this culture method. For example, the virus yield is reduced due to the limitation of the surface area of ​​the substrate in the adherent culture; Serum culture technology is an effective tool to elucidate the basic issues of cell generation, proliferation, differentiation and gene expression regulation, but even serum-free adherent culture is only suitable for experimental research, not for large-scale culture, and cannot meet the actual production of virus vaccines need

Method used

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  • Method for proliferating avian influenza virus by adopting MDCK cell line
  • Method for proliferating avian influenza virus by adopting MDCK cell line
  • Method for proliferating avian influenza virus by adopting MDCK cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] A kind of method adopting MDCK cell line propagation bird flu virus, comprises the steps:

[0044] Step 1. Mix DMEM, F12 and RPMI1640 evenly according to the ratio of 3:2:1, use this as the basal medium and add 12% FBS, revive and culture the adherent MDCK cells, after the cells grow to a dense monolayer, 0.25% EDTA-trypsin digestion, passage 6 times; gradually reduce the proportion of DMEM-F12-RPMI1640 medium containing 12% FBS, increase the proportion of serum-free DMEM-F12-RPMI1640 medium, and gradually reduce the serum content to 0%;

[0045] Step 2: Digest well-grown MDCK cells adapted to serum-free culture with 0.25% EDTA-trypsin, and then culture them on a shaking table at a speed of 50 r / min; after the cell growth rate is stable, subculture, gradually expand the culture, and gradually increase The shaker rotates until the MDCK cell line is acclimatized to a stable and proliferating serum-free full-suspension culture;

[0046] Step 3. Inoculate the above-mention...

Embodiment 2

[0051] A kind of method adopting MDCK cell line propagation bird flu virus, comprises the steps:

[0052] Step 1. Mix DMEM, F12 and RPMI1640 evenly in a ratio of 3:1:1, use this as the basal medium and add 10% FBS, revive and culture the adherent MDCK cells, and wait until the cells grow to a dense monolayer. 0.25% EDTA-trypsin digestion, passage 5 times; gradually reduce the proportion of DMEM-F12-RPMI1640 medium containing 10% FBS, increase the proportion of serum-free DMEM-F12-RPMI1640 medium, and gradually reduce the serum content from 10% to 0%;

[0053] Step 2. Digest well-grown MDCK cells adapted to serum-free culture with 0.25% EDTA-trypsin, and culture them on a shaking table at a speed of 40r / min; after the cell growth rate is stable, subculture, gradually expand the culture, and gradually increase The shaker rotates until the MDCK cell line is acclimatized to a stable and proliferating serum-free full-suspension culture;

[0054] Step 3. Inoculate the above-mentio...

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Abstract

The invention relates to a method for proliferating avian influenza virus by adopting an MDCK cell line. The method comprises the following steps: domesticating adherent MDCK cells into a serum-free full-suspension culture type MDCK cell line which is stably proliferated, inoculating a bioreactor with the MDCK cell line, completing primary cell culture, preparing avian influenza H5N1 subtype virusseed viruses for suspension MDCK cell culture, inoculating suspension MDCK cells with the avian influenza H5N1 subtype virus seed viruses, performing culturing, and separating and purifying virus particles. The cells obtained by domestication are stable in passage, good in cell morphology, high in quality, capable of keeping sensitivity and biological characteristics to viruses, high in virus multiplication capacity and capable of effectively multiplying avian influenza virus strains.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for propagating avian influenza virus using MDCK cell lines. Background technique [0002] Avian influenza (Bird Flu or Avian Influenza) is an acute infectious disease caused by a subtype of influenza A virus (also known as avian influenza virus), which seriously threatens the breeding industry and human health, and brings huge losses to the national economy. So far, no ideal treatment for bird flu has been found, and vaccination is still the most effective means to prevent the occurrence and spread of the disease. [0003] There are many problems in the traditional chicken embryo technology used in the preparation of bird flu vaccines. The production cycle is long, the operation is cumbersome, the workload is heavy, and it is easy to be polluted. Using cells as the medium is the current trend of vaccine preparation. Canine kidney (Madin-Darby canine kidney cells, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/071A61K39/145A61P31/16C12R1/93
CPCC12N7/00C12N5/0686A61K39/12A61P31/16C12N2760/16151C12N2500/90C12N2509/00C12N2760/16134A61K2039/5252A61K2039/5254A61K2039/5256A61K2039/53A61K2039/552
Inventor 施维松刘德城詹烜子郑彩丽卢少华孔凤英谢玉红
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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