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
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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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