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Preparation method for human diploid cell rabies vaccine virus solution

A technology of human diploid cells and rabies vaccine, which is applied in the biological field, can solve the problems of high host DNA residue, tumorigenic risk, DNA pollution, etc., and achieve increased harvest, shortened preparation process time, and high antigen content Effect

Inactive Publication Date: 2014-12-03
BEIJING MINHAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These vaccines all have various risk factors such as heterologous proteins, foreign factors, and DNA contamination
In addition, for primary cell culture, each batch of vaccines needs to be inspected for impurities (bacteria, mycoplasma, virus) from cultured organs, and the availability of animal organs is a factor that limits industrial production; while Vero cells are monkey-derived cells, and the host DNA residues are relatively high , there is a risk of tumorigenic

Method used

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  • Preparation method for human diploid cell rabies vaccine virus solution
  • Preparation method for human diploid cell rabies vaccine virus solution
  • Preparation method for human diploid cell rabies vaccine virus solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Preparation method of human diploid cell rabies vaccine virus solution (1)

[0032] Take the cryopreservation tube containing MRC-5 cells, put it into a 20°C water bath to melt quickly, and then add it to the pre-warmed to 25°C containing 20% ​​fetal bovine serum cell culture medium (BME culture medium, pH value is 6.90, osmotic pressure 300mosm / kg, without antibiotics), cultured at 37°C, and continuously expanded until the cells grew into a dense monolayer of cells. The monolayer cells were prepared as a cell suspension, and the rabies virus PM virus seed was inoculated at an MOI of 0.07, incubated in a water bath at 37°C for 15 minutes, then subpackaged and cultured at a ratio of 1:3 for 22 hours, and then adjusted to 35°C to continue culturing. After the virus cells grow into a single layer, first wash the cell surface with PBS solution, then digest the cells with 0.01% trypsin, digest the virus cells to prepare the final suspension of virus cells, divid...

Embodiment 2

[0038] Embodiment 2 Preparation method of human diploid cell rabies vaccine virus liquid (2)

[0039] Take the cryopreservation tube containing MRC-5 cells, put it into a 20°C water bath to thaw quickly, and then add it to the pre-warmed to 25°C containing 20% ​​fetal bovine serum cell culture medium (BME culture medium, pH value is 7.20, osmotic pressure 330mosm / kg, without antibiotics), cultured at 37°C, and continuously expanded to a certain number of cells. Prepare monolayer cells as a cell suspension, inoculate rabies virus 65 generation virus seeds at 0.06 MOI, incubate in a water bath at 37°C for 16 minutes, then subpackage and culture at a ratio of 1:3, incubate at 37°C for 24 hours and then adjust the temperature to 35°C to continue Culture, after the virus cells grow into a single layer, wash the cell surface with PBS solution first, then digest the cells with 0.01% trypsin, digest the virus cells to prepare the final suspension of virus cells, divide according to th...

Embodiment 3

[0045] Example 3 The preparation method of rabies virus harvest liquid is used to prevent large-scale industrial production of human diploid rabies vaccine

[0046] According to the preparation method of human diploid cell rabies vaccine harvest liquid, Roche bottle and cell factory are used to enlarge the production scale, and a single batch of 1000 people is produced in batches. It is confirmed that this method is suitable for large-scale production. Specific steps are as follows:

[0047] Prepare monolayer cells cultured in Roche flasks as cell suspension, inoculate rabies virus seeds at 0.1 MOI, absorb in water bath at 37°C for 16 minutes, then inoculate at 0.65-0.85ml / cm 2 Aliquot the virus cell suspension and culture in Roche flasks, culture at 37°C for 24 hours, then adjust the temperature to 35°C and continue to culture for 3 days. After the virus cells grow into a single layer, digest the infected cells with 0.01% trypsin solution to make the virus The final cell sus...

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Abstract

The invention provides a preparation method for human diploid cell rabies vaccine virus solution, and relates to the field of biotechnology. The preparation method comprises the step of inoculating rabies vaccine fixed virus PM-1503-3M strain in MRC-5 cell to generate the human diploid cell rabies vaccine virus solution. The method disclosed by the invention is simple in process and cost-saving; and the prepared virus solution is high in antigen content, high in virus valence and titer, suitable for large-scale industrialized production, and capable of meeting the needs of domestic and foreign markets on human diploid cell rabies vaccine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing virus liquid on human diploid cells by using PM strain of rabies virus. Background technique [0002] Rabies is endemic in more than 100 countries. More than 3.3 billion people worldwide live in rabies-endemic regions, especially in Asia and Africa. After humans are infected with rabies virus, once clinical symptoms appear, almost 100% of them die, and 99% of the deaths are caused by dog ​​rabies. In addition to domestic dogs, many carnivorous animals and bats can also transmit rabies virus to humans. Asia is a region with a high incidence of rabies, accounting for about 90% of human rabies deaths due to dog injuries worldwide. In 2000, about 37,000 people died from rabies among the 3.11 billion people living in 15 rabies-endemic countries in Asia. Most people die from exposure to latent rabies virus infection without treatment or vaccination. In 2009 and 2...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12R1/93
Inventor 左静高正伦刘海文方群季振涛郑森远张骞刘勇张芮铭代凡吴淞张雪戴会忠刘雅静王云达樊鹏
Owner BEIJING MINHAI BIOTECH
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