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Method for culturing MDCK (medin-darby canine kidney) cell proliferation recombination H5N1 subtype avian influenza virus

A bird flu virus and cell proliferation technology, applied in the direction of microorganism-based methods, animal cells, virus/bacteriophage, etc., can solve the problems of high cost, low cell density, chicken embryo contamination, etc., and achieve low chance of infection, cell The effect of large number and fast cell expansion

Active Publication Date: 2015-04-22
山东信得动物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, vaccine immunization is the main measure to prevent and control the outbreak of avian influenza at home and abroad. At present, most manufacturers in my country use SPF chicken embryos as the substrate to produce avian influenza vaccines. This process is very dependent on SPF chicken embryos; the virus liquid produced The titer is not high; the conditions in the cultivation process are not uniform enough, and there are large differences between batches; due to the inevitable contamination of chicken embryos, the endotoxin in the vaccine is high, which has a great impact on the immune effect; at the same time, the production of chicken embryos causes a large number of chickens. Embryo waste is also a great pollution to the ecological environment
[0003] Large-scale cultivation of animal cells with vectors in bioreactors to propagate avian influenza virus has become a new production method and has been popularized and applied. However, most manufacturers currently use microcarriers for virus propagation. Most of the microcarriers are produced by GE in the United States. At the same time, due to the limitation of the amount of microcarriers added per unit of culture medium, the cell density per unit volume is not high, so that it is impossible to obtain high-titer antigens

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] A method for cultivating MDCK cell proliferation recombinant H5N1 subtype avian influenza virus, comprising the following steps:

[0021] Seed cell supply: Use a 7-liter microcarrier bioreactor to expand and cultivate MDCK-α-2,3Gal cells, select healthy and well-growing cells, digest them with trypsin to make a cell suspension, and perform cell counting as a paper sheet Seed cells for vector bioreactors.

[0022] 1) Preparation of the sheet carrier: the polyester fiber paper carrier was cut into a wavy paper sheet with a length of 10 cm and a width of 2 cm.

[0023] 2) Paper carrier pretreatment: Step 1) Soak the obtained sheet carrier with 0.5mol / L sodium hydroxide solution for 16 hours, then wash it with water for injection until the pH is 7.6, let go of the water for injection, add PBS solution and soak until use.

[0024] 3) Sterilization and pre-cultivation of the bioreactor: Add the paper carrier treated in step 2) into the bioreactor, then add fresh PBS solutio...

Embodiment 2

[0030] Soak a circular paper carrier with a diameter of 2 cm in 1.0 mol / L sodium hydroxide solution for 12 hours, then wash it with water for injection until the pH is 7.4, drain the water for injection, add it to the bioreactor, and add PBS solution at the same time. After connecting all the pipelines of the bioreactor, perform steam sterilization at 121°C for 30 minutes. After the sterilization is completed, the tank temperature drops below 40°C, then release the PBS solution, add cell culture medium, and adjust the reactor temperature to 36°C for pre-cultivation. 16h. Select healthy and well-growing MDCK-α-2,3Gal cells after microcarrier expansion and culture, digest them with trypsin to make a cell suspension with a density of 1.9×107 cells / gram of paper carrier, and inoculate them after preculture In the bioreactor without abnormalities, adjust the reactor speed to 35rpm, and at the same time turn on the reactor control conditions, and adjust the culture parameters as fol...

Embodiment 3

[0033] Soak a circular paper carrier with a diameter of about 2 cm in 0.5 mol / L sodium hydroxide solution for 12 hours, then wash it with water for injection until the pH is 7.6, drain the water for injection, add it to the bioreactor, and add PBS solution at the same time. After connecting all the pipelines of the bioreactor, perform steam sterilization at 121°C for 30 minutes. After the sterilization is completed, the tank temperature drops below 40°C, then release the PBS solution, add cell culture medium, and adjust the reactor temperature to 36°C for pre-cultivation. 24h. Select healthy and well-growing MDCK-α-2,3Gal cells after microcarrier expansion and culture, digest them with trypsin to make a cell suspension with a density of 2.0×107 cells / gram of paper carrier, and inoculate them after preculture In the bioreactor without abnormalities, adjust the reactor speed to 40rpm, and at the same time turn on the reactor control conditions, and adjust the culture parameters ...

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Abstract

The invention provides a method for culturing MDCK (medin-darby canine kidney) cell proliferation recombinant H5N1 subtype avian influenza virus comprises the following steps: preparing a chip carrier, pretreating a paper scrap carrier, sterilizing a bioreactor, carrying out preculture, inoculating cells, adsorbing on the paper scrap carrier, continuously culturing cells, inoculating avian influenza virus and harvesting influenza virus bulk. The method for culturing the MDCK cell proliferation recombinant H5N1 subtype avian influenza virus has the beneficial effects that carrier cost is low, hematic coagulating rate of obtained influenza virus bulk is high, the paper scrap carrier is adopted for carrying out bioreactor large-scale culture of MDCK cell proliferation recombinant H5N1 subtype avian influenza virus, large-scale culture can be realized more easily, occupying space of a production line is small, production efficiency is high, and the adopted equipment has the advantages of strong stability, good repeatability and the like.

Description

technical field [0001] The invention relates to a method for avian influenza virus propagation, in particular to a method for large-scale cultivation of MDCK cells in a paper carrier bioreactor to propagate avian influenza virus. Background technique [0002] Avian influenza (AI) is a severe infectious disease of poultry caused by type A avian influenza virus. At present, vaccine immunization is the main measure to prevent and control avian influenza outbreaks at home and abroad. At present, most manufacturers in my country use SPF chicken embryos as the substrate to produce avian influenza vaccines. This process is very dependent on SPF chicken embryos; the virus liquid produced The titer is not high; the conditions in the cultivation process are not uniform enough, and there are large differences between batches; due to the inevitable contamination of chicken embryos, the endotoxin in the vaccine is high, which has a great impact on the immune effect; at the same time, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/071C12R1/93
Inventor 李朝阳刘志亮裴香玲李明义宋桂才乔彦良马广斌王富猛朱元苍
Owner 山东信得动物疫苗有限公司
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