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Human diploid cell rabies vaccine virus seed and preparation method thereof

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

Active Publication Date: 2013-05-01
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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Human diploid cells (KMB17) were cultured and amplified at 35° C. to 37° C. with a cell nutrient solution composed of Eagle solution, milk protein and 2% to 15% newborn bovine serum. When the cells grow into a dense monolayer, they are digested with trypsin-EDTA, the cells are collected, and CTN-1V 5 The strain is inoculated in human diploid cells (KMB17) by the suspension cell inoculation method at a dose of 0.005-1.0 MOI, cultured at 32°C-37°C, pH 7.2-8.0, and the virus is harvested after 2-20 days of culture, that is, fixed rabies virus. CTN-DK1, and continued passage in the same way. When subcultured to CTN-DK20, the virus with higher titer was screened by the final dilution method and continued for 9 passages, named CTN-DK29, and then screened by the final dilution method of CTN-DK29 and passed for 4 generations, named CTN -DK33, and then selected by the final dilution method with CTN-DK33 and passed to 47 passages. CTN-1V 5 The strain was continuously passaged ...

Embodiment 2

[0022] Using the suspension cell inoculation method, take the cells that have grown into a single layer, digest them with trypsin routinely, collect the cells, and then suspend them in a growth medium (Eagle lactalbumin culture medium containing 10% to 15% newborn bovine serum) at an appropriate ratio cell. Inoculate the virus in suspension cells, culture at 37°C until the cells grow into a monolayer, remove the original culture medium, replace with maintenance medium (Eagle lactalbumin culture medium containing 2% to 5% newborn calf serum), and place at 32°C, Cultivate at 33°C, 34°C, 35°C, 36°C, 37°C, and harvest the virus liquid after 5-7 days. The harvested virus liquid was detected by rabies virus titer titration test (LD50), and the results are shown in Table 1.

[0023] The virus titer (1gLD50 / ml) of table 1 virus kind in different cultivation temperature in human diploid cell (KMB17)

[0024]

[0025] The above data were tested by T test, 0.2

Embodiment 3

[0027] In order to determine the optimal inoculum amount of virus species, experiments were carried out on different multiplicity of infection (MOI) of rabies virus fixed virus CTN-DK strain infecting human diploid cells (KMB17). The method is to make the KMB-17 cells growing in the logarithmic phase into a cell suspension and count them. Inoculate the suspension cells with the titrated virus at 1.0, 0.5, 0.1, 0.05, 0.01, 0.005 MOI, culture at a suitable temperature, and harvest the virus liquid after culturing for a certain period of time. The harvested virus liquid was detected by rabies virus titer titration test (LD50), and the results are shown in Table 2.

[0028] The virus titer (1gLD50 / ml) of table 2 virus kind different MOI infection human diploid cells (KMB17)

[0029]

[0030] The results showed that there was no significant difference in the production of cells infected with different numbers of viruses. Therefore, it is more appropriate to use 0.005-1.0 MOI to ...

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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 using human diploid cells (KMB17) to produce vaccine virus seeds for preventing human rabies and a preparation method thereof. Background technique [0002] Rabies is a disease caused by the rabies virus, once the onset, 100% fatal. In recent years, the rabies epidemic in my country has continued to rise, and the incidence rate is now the second in the world, second only to India. The number of deaths from rabies ranks first among the 37 legally notifiable infectious diseases. Rabies cannot be cured so far but can be effectively prevented, so the prevention of rabies in our country is particularly important. [0003] At present, the culture substrate cells used in the production of rabies vaccine 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 cannot ...

Claims

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Application Information

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Patent Type & Authority Patents(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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