A kind of preparation method of virus stock solution

A virus stock solution and virus technology, applied in the field of virus stock solution preparation, can solve the problems affecting the infection degree of heterotropic mouse leukemia virus, low virus titer, and low proliferation efficiency of host cells, so as to improve growth rate and proliferation efficiency , high virus titer, and the effect of promoting the amplification of the virus

Active Publication Date: 2021-11-30
苏州良辰生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, during the use of polybrene, polybrene has a toxic effect on the host cell in the process of promoting heterophile murine leukemia virus to enter the cell and replicate, which makes the proliferation efficiency of the host cell itself not high, thus affecting the heterophilic murine leukemia virus. The degree of infection with murine leukemia virus results in lower titers of amplified virus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Inoculate CHO seed cells into 10 mL of MEM medium containing 10% fetal bovine serum by volume for culture, freeze storage and build a bank. Take a cell line from the working cell bank and inoculate until 10 mL of MEM medium containing volume percentage is added. 55cm of the MEM medium with a percentage concentration of 10% fetal bovine serum 2 Continue culturing in the petri dish;

[0030] (2) After host cells grow on more than 80% of the surface of the culture dish, wash it twice with MEM serum-free medium;

[0031] (3) Dilute the dextran with 0.01M phosphate buffered saline (1×PBS), and thoroughly mix it with heterotropic mouse leukemia virus to obtain a mixture, the concentration of dextran in the mixture is 15ug / ml, the volume percent concentration of heterotropic murine leukemia virus is 0.1%, and then 1mL of the mixed solution is added to the above-mentioned culture dish for adsorption infection. During the infection process, the culture medium is slightly s...

Embodiment 2

[0034] (1) Inoculate CHO seed cells into 10 mL of MEM medium containing 10% fetal bovine serum by volume for culture, freeze storage and build a bank. Take a cell line from the working cell bank and inoculate until 10 mL of MEM medium containing volume percentage is added. 55cm of the MEM medium with a percentage concentration of 10% fetal bovine serum 2 Continue culturing in the petri dish;

[0035] (2) After host cells grow on more than 80% of the surface of the culture dish, wash it twice with MEM serum-free medium;

[0036] (3) Dilute the dextran with 0.01M phosphate buffered saline (1×PBS), and thoroughly mix it with heterotropic mouse leukemia virus to obtain a mixture, the concentration of dextran in the mixture is 20ug / ml, the volume percent concentration of heterotropic murine leukemia virus is 0.1%, and then 1mL of the mixed solution is added to the above-mentioned culture dish for adsorption infection. During the infection process, the culture medium is slightly s...

Embodiment 3

[0039] (1) Inoculate CHO seed cells into 10 mL of MEM medium containing 10% fetal bovine serum by volume for culture, freeze storage and build a bank. Take a cell line from the working cell bank and inoculate until 10 mL of MEM medium containing volume percentage is added. 55cm of the MEM medium with a percentage concentration of 10% fetal bovine serum 2 Continue culturing in the petri dish;

[0040] (2) After host cells grow on more than 80% of the surface of the culture dish, wash it twice with MEM serum-free medium;

[0041] (3) Dilute dextran with 0.01M phosphate buffered saline (1×PBS), and fully mix it with heterotropic mouse leukemia virus into the mixture. The concentration of dextran in the mixture is 30ug / ml, the volume percent concentration of heterotropic murine leukemia virus is 0.1%, and then 1mL of the mixed solution is added to the above-mentioned culture dish for adsorption infection. During the infection process, the culture medium is slightly shaken so tha...

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Abstract

The invention relates to a preparation method of a virus stock solution. The host cells are cleaned with a serum-free medium; dextran, phosphate buffer and virus are prepared into a mixed solution; the mixed solution is added to the treated host cells Carry out adsorption infection; culture the treated host cells with a medium containing fetal bovine serum; freeze and thaw the treated host cells to release the virus, and then centrifuge to obtain the virus stock solution. The present invention replaces polybrene by adopting dextran, because dextran itself is a kind of cationic polymer, it is combined with the negatively charged virus and is close to the cell membrane to be taken up, and has no toxic effect on the cell itself, fundamentally In fact, it can improve the growth rate and proliferation efficiency of host cells, and play a significant role in promoting the amplification of viruses, so that the virus titer of the prepared virus stock solution is high.

Description

technical field [0001] The invention relates to a preparation method of virus stock solution. Background technique [0002] The virus stock solution can be used to prepare vaccines, and can also be added as an indicator virus in the virus inactivation verification experiment. When used as an indicator virus in the virus inactivation verification experiment, the greater the virus titer of the virus stock solution, the more virus that needs to be added. Fewer stock solutions can reduce the cost of virus inactivation verification experiments. [0003] In the prior art, the stock solution of heterotropic murine leukemia virus is usually inoculated into host cells with a solution containing heterotropic murine leukemia virus, and co-cultivated. Polybrene needs to be added to the culture medium to improve heterotropic The infection efficiency of mouse leukemia virus entering host cells (the main function of polybrene is to improve the efficiency of retrovirus infection of cells, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/93
CPCC12N7/00C12N2740/13051
Inventor 汪涛
Owner 苏州良辰生物医药科技有限公司
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