A method for suspending and culturing infectious bronchitis virus with passaged cell lines
A technology for bronchitis and subculture cells, which is applied in the direction of viruses, virus/bacteriophage, biochemical equipment and methods, etc. It can solve the problems of inability to realize large-scale full suspension culture and low virus titer, so as to reduce the cost of cultivation, virus titer, etc. High degree and good immunogenicity
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Embodiment 1
[0029] Example 1: Effects of Different Inoculation Doses on the Proliferation of Chicken Infectious Bronchitis Virus in EB66 Cells
[0030] The virus used in the embodiment of the present invention 1 is that chicken infectious bronchitis virus is IBV (M41 strain), and the poison price is 10 7.3 EID 50 / 0.1mL, identified, kept and supplied by Zhaoqing Dahuanong Biopharmaceutical Co., Ltd.
[0031] The cultivation method of the embodiment of the present invention 1 chicken infectious bronchitis virus (M41 strain) is as follows:
[0032] Step 1: Take out the frozen EB66 cell line from the liquid nitrogen tank and place it in a 37°C water bath. Add the thawed cell line to about 30 mL of the medium of the EB66 cell line (Jinshun Biological Product No. CD210), and pass through a centrifuge Centrifuge at 300g for 10min, remove the supernatant, resuspend the cells in 15mL culture medium derived from duck embryo cells in a 125mL Erlenmeyer shaker flask, and place the culture medium a...
Embodiment 2
[0041] Example 2: Effects of different TPCK supplementation strategies on the proliferation of chicken infectious bronchitis virus
[0042] The virus source used in Example 2 of the present invention is the EID harvested in Example 1 50 Avian infectious bronchitis virus (M41 strain) at the highest titer.
[0043] The culture method of the embodiment of the present invention 2 infectious bronchitis virus is as follows:
[0044] Step 1: 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; wherein, the generation of EB66 cells inoculated with poisoned cells is limited to the cells after recovery from the working bank. Within 3-30 generations;
[0045] Step 2: When cells grow to a density of 8 x 10 in step 1 6 / mL~10×10 6 / mL, carry out the inoculation of chicken infectious bronchitis virus (M41 strain) according to the inoculation a...
Embodiment 3
[0050] Embodiment 3: EID of chicken infectious bronchitis virus at different harvest times in EB66 cell culture 50 Compare
[0051] The source of virus used in Example 3 of the present invention is the EID harvested in Example 2 50 Avian infectious bronchitis virus (M41 strain) at the highest titer.
[0052] The cultivation method of the embodiment of the present invention 3 infectious bronchitis 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 passaged cell line derived from duck embryo cells The generation of infected cells is limited to within 3-30 generations after recovery of the cell working bank;
[0054] 2. When the cells in step 1 grow to a density of 8×10 6 / mL~10×10 6 At the time of / mL, it is 0.01MOI to carry out the inoculation ...
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