Human diploid cell rabies vaccine virus seed and preparation method thereof

A technology for human diploid cells and rabies vaccine, applied in the biological field, can solve the problems of high vaccine cost, low virus titer, high price, etc., and achieve improved safety, major social and economic benefits, and good immunogenicity. Effect

Active Publication Date: 2011-06-15
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The human diploid cell rabies vaccine produced abroad, such as the diploid vaccine produced by the Wyeth factory in the United States, the Merieux Institute in France and the Behring factory in Germany, has been confirmed to have good immunogenicity and safety, but the human diploid cell Cultivation of rabies virus has disadvantages such as relatively low virus titer, high vaccine cost and high price, making it difficult to be widely used

Method used

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  • Human diploid cell rabies vaccine virus seed and preparation method thereof
  • Human diploid cell rabies vaccine virus seed and preparation method thereof
  • Human diploid cell rabies vaccine virus seed and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Human diploid cells (KMB17) were cultured and expanded at 35°C to 37°C with a cell nutrient solution consisting of Eagle's solution, milk protein and 2%-15% newborn bovine serum. When the cells grew into a dense monolayer, they were digested with trypsin-EDTA, the cells were collected, and CTN-1V 5 The strain was inoculated into human diploid cells (KMB17) by the suspension cell inoculation method at a dose of 0.005 to 1.0 MOI, cultured at 32 ° C to 37 ° C, pH 7.2 to 8.0, and the virus was harvested after culturing for 2 to 20 days. CTN-DK1, and continued passage in the same way. when passaged

[0021] At the time of CTN-DK20, the virus with higher titer was screened by the terminal dilution method and continued to be passaged for 9 generations, named CTN-DK29, and then screened by the terminal dilution method with CTN-DK29 and passed down for 4 generations, named CTN-DK33 , and then screened by terminal dilution method with CTN-DK33 and passed to 47 generations. CTN...

Embodiment 2

[0023] Using the suspension cell inoculation method, take the cells that have grown into a monolayer, routinely digest them with trypsin, collect the cells, and then suspend them in a growth medium (Eagle lactalbumin culture medium containing 10% to 15% newborn calf serum) in an appropriate proportion cell. The virus was inoculated into the suspension cells, cultured at 37°C until the cells grew into a monolayer, the original culture medium was removed, and the maintenance medium (Eagle lactalbumin medium containing 2% to 5% newborn calf serum) was replaced. Incubate at 33°C, 34°C, 35°C, 36°C, and 37°C, and harvest the virus solution in 5 to 7 days. The harvested virus liquid was detected by rabies virus titer titration test (LD50). The results are shown in Table 1.

[0024] Table 1 Virus titers (lgLD50 / ml) of virus species at different culture temperatures in human diploid cells (KMB17)

[0025]

[0026] The above data were tested by T test, 0.2

Embodiment 3

[0028] In order to determine the optimal inoculation amount of the virus species, experiments were carried out to infect human diploid cells (KMB17) with different multiplicity of infection (MOI) of the immobilized rabies virus CTN-DK strain. The method is to make a cell suspension of KMB-17 cells grown in log phase and count them. The titrated virus was inoculated into suspension cells at MOI of 1.0, 0.5, 0.1, 0.05, 0.01, 0.005, cultured at a suitable temperature, and the virus liquid was harvested after culturing for a certain period of time. The harvested virus liquid was detected by rabies virus titer titration test (LD50). The results are shown in Table 2.

[0029]Table 2 Virus titers (lgLD50 / ml) of human diploid cells (KMB17) infected with different MOIs of virus species

[0030]

[0031] The results showed that there was no significant difference in the yield of cells infected with different virus numbers. Therefore, it is more appropriate to use 0.005-1.0 MOI to i...

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Abstract

The invention relates to the field of biotechnology, in particular to a virus seed for producing vaccines for preventing human rabies by utilizing human diploid cells (KMB17) and a preparation method thereof. In the invention, a rabies fixed virus CTN-1V5 strain is continuously subcultured in the human diploid cells (KMB17), and a terminal dilution method is used for screening viruses with highertiter, thereby obtaining a rabies virus strain which is suitable for the human diploid cells (KMB17) and has good immunogenicity and heredity stability, and culturing a rabies vaccine virus seed (CTN-DK strain) capable of efficiently reproducing in the human diploid cells (KMB17). By using the virus seed for producing a human diploid cell (KMB17) rabies vaccine, the risk caused by residual heterogonous DNA (deoxyribonucleic acid) in the vaccine which is currently used at home can be effectively avoided, and the safety and practicability of the rabies vaccine in China are further improved, thus the invention has great social and economic benefits.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the use of human diploid cells (KMB17) to produce a vaccine virus seed for preventing human rabies and a preparation method thereof. Background technique [0002] Rabies is a disease caused by the rabies virus, and once it develops, 100% of people die. In recent years, the rabies epidemic in my country has continued to rise, and the incidence rate now ranks second in the world, second only to India. The death toll from rabies ranks first among the 37 notifiable infectious diseases. Rabies cannot be cured but can be effectively prevented. Therefore, the prevention of rabies in my country is particularly important. [0003] At present, the culture substrate cells used in the production of rabies vaccines in the world mainly include animal-derived cells such as hamster kidney cells, chicken embryo cells and African green monkey kidney cells (Vero cells). Animal-derived primary cells c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 毛子安黄海鹰姜立民李永学陈秋红林晓波高磊严宵周康凤
Owner ZHEJIANG PUKANG BIOTECH
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