Method for suspension culture of infectious bronchitis virus by continuous cell line

A bronchitis and suspension culture technology, applied in the direction of viruses, viruses/phages, positive-sense single-stranded RNA viruses, etc., can solve the problems of inability to achieve large-scale full suspension culture and low virus titers, and reduce the cost of culture and the reduction of virus droplets. High degree of efficacy and good immunogenicity

Active Publication Date: 2018-07-20
ZHAOQING INST OF BIOTECHNOLOGY CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it has been reported that the passaged cell lines Vero cell line and BHK-21 cell line are used to culture chicken infectious bronchitis virus, but they cannot really replace the traditional ones because of the low titer of the cultured virus or the inability to realize large-scale full suspension culture. chicken embryo inoculation

Method used

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  • Method for suspension culture of infectious bronchitis virus by continuous cell line
  • Method for suspension culture of infectious bronchitis virus by continuous cell line
  • Method for suspension culture of infectious bronchitis virus by continuous cell line

Examples

Experimental program
Comparison scheme
Effect test

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, place it in a 37°C water bath, add the thawed cell line to about 30mL of CD EB66210 (Jin Shun biological product number DP210) medium, and centrifuge Centrifuge at 300g for 10min, remove the supernatant, resuspend the cells in 15mL of CD EB66210 medium in a 125mL Erlenmeyer shaker flask, and place the medium at 37°C, 5% CO 2 In the incubator, set the rotation speed of th...

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 inoculated cells is limited to 3 days after recovery of the cell working bank. - within 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 amount of 0.01MOI, th...

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 generation of EB66 inoculated 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 of infectious bronchitis virus by the amount of...

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Abstract

The invention provides a method for suspension culture of an infectious bronchitis virus by a continuous cell line. The method comprises the following steps that 1, EB66 cells are taken for recovery and secondary culture; 2, the EB66 cells obtained in the step 1 are subjected to infectious bronchitis virus inoculation culture; 3, the EB66 cells after the virus inoculation are sampled every 6 to 12h; virus EID50 is determined; when the virus EID50 reaches the highest value, the virus is harvested and stored; the cultured infectious bronchitis virus is obtained. The EB66 cells are used as a culture medium of the infectious bronchitis virus; the EB66 cells providing a full suspension continuous cell line is used for performing efficient virus production; the culture titer of the infectious bronchitis virus is effectively improved, so that the large-scale culture of the infectious bronchitis virus vaccine is realized; the virus culture cost is reduced.

Description

technical field [0001] The invention relates to a full-suspension culture method for chicken infectious bronchitis virus, in particular to a method for full-suspension culture of chicken infectious bronchitis virus by using subcultured cell lines, and belongs to the technical field of veterinary biological products. Background technique [0002] Chicken infectious bronchitis (Avian Infectious Bronchitis, IB) is an acute, highly contagious viral infectious disease, mainly caused by chicken infectious bronchitis virus (Infectious Bronchitis Virus, IBV). It is one of the major infectious diseases that seriously endanger the poultry industry in the world, and is listed as a B-type disease by the World Organization for Animal Health (OIE). The disease mainly affects the respiratory system, urinary system and digestive system of chickens, showing extensive tissue tropism and high genetic variability, which can lead to different degrees of disease and mortality in chickens of diffe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02
CPCC12N7/00C12N2770/20051
Inventor 李延鹏陈瑞爱罗顺蔡仕君温良海罗琼张晓楠
Owner ZHAOQING INST OF BIOTECHNOLOGY CO LTD
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