Method for producing rabies viruses by suspension culture of BHK21 cells

Abstract

The invention provides a method for producing rabies viruses by the suspension culture of BHK21 cells, which comprises a step of performing suspension culture of BHK21 cells in bioreactor containing a BHK21 cell culture medium, wherein the conditions for the suspension culture in the step include a temperature of 32 to 36 DEG C, a pH value of 7.0 to 8.0 and a dissolved oxygen concentration of 30 to 50 percent; and the BHK21 cell culture medium comprises the components shown in a table 1. The method overcomes the biases of the prior art and realizes the production of the rabies viruses by the suspension culture of BHK21 cells in the bioreactor. The obtained rabies viruses can be used for producing rabies vaccine. Due to the automatic culture environment parameter control of the bioreactor, the cells grow and the viruses propagate in more favorable environments, the virus titer is improved and the large-scale automatic continuous production can be realized.

Description

technical field [0001] The invention relates to a method for culturing cells to produce virus, in particular to a method for suspending culture BHK21 cells to produce rabies virus. Background technique [0002] Rabies is a zoonotic infectious disease caused by Rabies Virus, which has the highest fatality rate (almost 100%) in the world. Mammals, especially dogs and cats, are susceptible. And cats and dogs as hosts of rabies virus can also transmit rabies virus to humans. Therefore, the epidemiological control of rabies mainly relies on expanding the coverage of animal vaccination and immunization; especially with the increase in the number of domestic pets in recent years, it is even more necessary to strengthen the strict management of pet immunization such as dogs and cats. High-quality veterinary rabies vaccines are the basis for successful immunization; and to prepare rabies vaccines, especially veterinary rabies vaccines, large-scale production of rabies virus species...

AI Technical Summary

Problems solved by technology

[0006] 1. When large-scale spinner bottles are used to culture BHK21 cells, hundreds of spinner bottles will be used, but each spinner bottle is an independent culture unit, and the state of cells and virus yields cultured in each spinner bottle are different. Therefore, the quantity and quality of the rabies virus produced cannot be effectively controlled, and the quality of the vaccine produced by the rabies virus cannot be effectively controlled.

[0007] 2. BHK21 cells are cultured in spinner bottles. Due to the adherent growth characteristics of BHK21 cells, BHK21 cells need to be digested for passage; in addition, after inoculating virus, adding and replacing virus maintenance solution, adding NaHCO 3 Adjusting the pH value and finally harvesting the culture requires multiple openings of the spinner bottle, with a high risk of contamination

And each spin bottle requires manual aseptic operation, the work is labor-intensive, human errors are large, and automation cannot be realized

[0009] 4. BHK21 cells cultured in spinner bottles cannot be sampled and counted at any time. It is difficult to accurately determine the amount of virus inoculated when inoculated with rabies virus

[0010] In summary, the existing method for producing rabies virus by cultivating BHK21 cells in spinner bottles is low in automation and labor-intensive, and the cell culture environment in each spinner bottle is uncontrollable, thus causing BHK21 cells, and the rabies virus grown in BHK21 cells, And the quality of the final rabies vaccine is unstable, and the product batches vary greatly

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