Full-suspension culture method for avian influenza virus

An avian influenza virus and a culture method technology are applied in the field of veterinary biological products, and can solve the problems of not being able to produce avian influenza vaccines in large quantities, chicken embryos being easily affected by avian influenza epidemics, and being easily brought into virus liquid, etc. Risks of introducing foreign viruses, good economic benefits and application prospects, and the effect of reducing immune side effects in chickens

Active Publication Date: 2018-04-13
ZHAOQING INST OF BIOTECHNOLOGY CO LTD +2
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Problems solved by technology

Restricted by these factors, the chicken embryos without specific pathogens (Spedfic Pathogen Free, SPF) have small output and high cost, which often cannot meet the needs of mass production of avian influenza vaccines
In addition, there are still many problems in the cultivation of chicken embryos. The quality of different batches of chicken embryos is often different, and it is difficult to control the quality. Chicken embryos of different quality will have different side effects on virus culture, and the source of chicken embryos is easily affected by poultry. The impact of the influenza epidemic; in addition, chicken embryos are the natural host of many bacteria, and are susceptible to bacterial infection in the environment. Some researchers have pointed out that the potential pollution brought by maternal inheritance to chicken embryos is more than artificial operation in a sterile environment in a GMP workshop. The resulting pollution rate is much higher, and this is where it is difficult to control in the chicken embryo culture process
In addition, the allantoic fluid of chicken embryos contains a large amount of animal-derived protein, which is easily brought into the virus fluid when the virus is extracted, resulting in a large amount of animal-derived protein in the finished vaccine, causing allergic reactions and other side effects
[0004] At present, the technology of using the passaged cell line MDCK cells as a culture substrate to produce avian influenza virus vaccines has become more and more mature, and has even reached the level of full-suspension mass production. Although MDCK cells can be cultured in high-density suspension, the cultured avian influenza virus Virus titer can reach chicken embryo level, but immunogenicity and animal side effects have always been technical barriers to using MDCK cells to produce avian influenza virus vaccines
Compared with chicken embryos, at the same hemagglutination value, the level of antibodies produced by the avian influenza virus produced by MDCK cells after immunizing chickens is low, and the side effects are severe, and MDCK cells can induce tumors in nude mice, and there is a tumorigenic effect. Risk, leading to lower safety of vaccine use, which is a fatal shortcoming for the safety of vaccines

Method used

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  • Full-suspension culture method for avian influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-4

[0030] The HA titer detection method of embodiment 1-4 gained avian influenza virus is as follows:

[0031] On a 96-well microplate, add 50 μL of normal saline to each well from left to right. And add 50 μL of virus solution to the first well on the left side, after mixing evenly, suck 50 μL to the second well, sequentially perform doubling dilution to the 11th well, draw and discard 50 μL, the 12th well is the red blood cell control; from right to On the left, add 50 μL of 1% chicken red blood cell suspension to each well in turn, vibrate on a shaker, and observe the results after standing at room temperature for 25-30 minutes. All red blood cells agglutinate, sink to the bottom of the well, and spread in a net shape, that is, 100% agglutination (++++), and red blood cells sink to the bottom of the well and appear as dots, which means no agglutination (-).

Embodiment 1

[0033] The sources of materials used in Embodiment 1 of the present invention are as follows:

[0034] 1. Virus: Type A avian influenza virus A / chicken / Gμangdong / DA / 2014 (H9N2) strain (DA strain for short), identified, kept and supplied by Zhaoqing Dahuanong Biological Pharmaceutical Co., Ltd.

[0035] 2. Cells: EB66 cells, a full-suspension cell line, were provided by Gansu Jianshun Biotechnology Co., Ltd.

[0036] 3. Serum-free, chemically defined medium: CD EB66 (DP210) medium, provided by Gansu Jianshun Biotechnology Co., Ltd.

[0037] The cultivation method of the embodiment of the present invention H9 subtype avian influenza virus is as follows:

[0038] 1. Take out the frozen EB66 cell line from the liquid nitrogen tank, put it in a 37°C water bath to melt, add it to about 30mL of CDEB66210 (Jianshun biological product number DP210) medium, centrifuge at 300g for 10min, and remove the supernatant , use a 125mL Erlenmeyer flask to resuspend the cells in 15mL of CD EB6621...

Embodiment 2

[0047] Example 2: Effects of Different TPCK Supplementary Strategies on the Proliferation of Avian Influenza Viruses

[0048] The source of materials used in Embodiment 2 of the present invention is as follows:

[0049] 1. Virus: the avian influenza virus when the HA titer harvested in Example 1 was the highest.

[0050] 2. Cells: EB66 cells, a full-suspension cell line, were provided by Gansu Jianshun Biotechnology Co., Ltd.

[0051] 3. Serum-free, chemically defined medium: CD EB66 (DP210) medium, provided by Gansu Jianshun Biotechnology Co., Ltd.

[0052] The cultivation method of the embodiment of the present invention H9 subtype avian influenza virus is as follows:

[0053] 1. Passage and culture the EB66 cells. The cells used in this implementation case are still the cells that continue to be passaged in the implementation case 1. The passage method and cell culture conditions are the same as in Example 1; among them, the generation of EB66 inoculated cells is limited ...

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Abstract

The invention discloses a full-suspension culture method for avian influenza virus. The full-suspension culture method comprises the following steps: step 1, taking EB66 cells for growth culture; step2, when the density of the EB66 cells grows to be suitable for virus inoculation, utilizing a fed-batch fluid-replacement virus inoculation technology to inoculate avian influenza virus into the EB66cells and performing virus multiplication culture; step 3, 24h after virus inoculation, sampling every 12h to determine HV titer of the virus and obtaining and storing the virus when the virus HA titer reaches to the maximum to obtain cultured avian influenza virus. According to the full-suspension culture method disclosed by the invention, the EB66 cells are utilized to perform avian influenza virus culture, so that the defect that a lot of miscellaneous protein is prone to being introduced into when a traditional chick embryo culture technology is utilized for production is overcome, and occurrence of chicken immunity side reaction is effectively reduced; meanwhile, titer and purity of the cultured avian influenza virus are improved; furthermore, production quality of the avian influenza vaccine is improved.

Description

technical field [0001] The invention relates to a full-suspension culture method of avian influenza virus, in particular to a method for the suspension culture of avian influenza virus by a full-suspension subculture cell line, belonging to the technical field of veterinary biological products. Background technique [0002] At present, most of the avian influenza vaccines on the market are produced through traditional chicken embryo culture, and chicken embryos have been used in the production of avian influenza virus at home and abroad for many years. The process is simple and mature. The main process of the preparation process is as follows: firstly, the seed venom is inoculated into the allantoic fluid of the chicken embryo. As a whole egg cell, the chicken embryo will not produce antibodies to the inoculated virus due to the low degree of tissue differentiation. It is conducive to the replication of the virus; after the virus is cultivated in the chicken embryo for a cer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02C12N5/0735C12N5/02
CPCC12N5/0606C12N7/00C12N2760/16151
Inventor 蔡仕君陈瑞爱罗顺李延鹏温良海张晓楠
Owner ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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