Rabies virus SNK-CTN strain and application thereof

A technology of rabies virus and virus strains, which is applied in antiviral agents, viruses/bacteriophages, medical preparations containing active ingredients, etc., can solve the problems of difficult vaccine extraction, unstable production, and many impurities, and achieve good protection effect. Reduce the steps of extraction and purification, the effect of simple vaccine extraction

Inactive Publication Date: 2015-02-18
SHINAIKE JIANGSU BIOLOGICAL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The inventor has found through research that the human diploid cell rabies virus strain in the prior art has many problems when preparing vaccines, such as: low titer, unstable yield, difficult vaccine extraction, and many impurities. The diploid cell rabies virus strain was screened, and a diploid cell rabies virus strain with excellent performance was obtained and used in production, achieving good technical results

Method used

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  • Rabies virus SNK-CTN strain and application thereof
  • Rabies virus SNK-CTN strain and application thereof
  • Rabies virus SNK-CTN strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1 Adaptation of rabies virus CTN strain on MRC-5 cells

[0052] Take the cryopreservation tube containing MRC-5 cells, put it into a 40°C water bath to melt quickly, then add it to the MEM cell culture medium pre-warmed to 37°C, culture it at 37°C, and grow into a single layer after 3-4 days After digestion with 0.25 trypsin, inoculate CTN-1V5 strain suspension at 0.01-1.0 MOI dose, culture at 37°C for 2-3 days, replace with maintenance solution containing 2-4% bovine serum, and cultivate at 33-35°C for 3-5 days The virus liquid was harvested, frozen at -70°C, and prepared into a virus suspension.

[0053] According to the above method, the harvested virus suspension was continuously uploaded to MRC-5 cells for 30 generations, and finally a rabies virus SNK-CTN strain with good adaptability to MRC-5 cells was obtained.

[0054] The rabies virus SNK-CTN strain that the present invention obtains carries out following observation and experiment:

[0055] (1) S...

Embodiment 2

[0057] Example 2 Application of SNK-CTN strain to prepare diploid cell rabies vaccine

[0058] Take human embryonic lung diploid cells (MRC-5) cultured in monolayer cell flasks, digest the cells with 0.25% trypsin, amplify the cells to the cell factory or bioreactor, add MEM cell culture medium, and the cells The medium was inoculated with SNK-CTN rabies virus at 0.05-0.IMOI, cultured at 37°C for 3 days, discarded the culture medium and replaced with maintenance medium. Place the culture at 35°C for 3 to 15 days, and harvest the virus liquid for 2 to 3 times according to the pathological changes of the cells in the cell factory; harvest the virus multiple times according to the indicators monitored by the reactor and the pathological changes of the cells as the virus harvest liquid.

[0059] The harvest liquid was inactivated by adding 1:4000 β-propiolactone under sterile conditions for 24 hours. After passing the inactivation test, the inactivated rabies virus liquid is conc...

example 3

[0062] Example 3 SNK-CTN strain prepares the quality inspection of diploid cell rabies vaccine

[0063] 1) Determination of virus titer

[0064] 10-fold serial dilution of the sample, intracerebral challenge of mice, each 0.03ml per dilution intracerebral inoculation of at least 6 mice weighing 11-13g, observed for 14 days, the virus titer should not be less than 7.0lg LD 50 / ml.

[0065] 2) Determination of antigen content

[0066] The purified anti-rabies virus polyclonal antibody was used to coat the enzyme-linked reaction plate and labeled horseradish peroxidase (HRP), and the double-antibody sandwich principle was used to quantitatively detect the antigen content in the rabies vaccine by using a two-step reaction mode. , should not be less than 0.4IU / dose.

[0067] Antigen content of human rabies vaccine and results of virus titer of corresponding virus harvest fluid

[0068] Test items

SNK-CTN Harvest Solution 1

SNK-CTN Harvest Solution 2

SNK-CT...

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Abstract

The invention relates to the field of microbes and biological pharmacy, and particularly relates to an adapted strain of a rabies virus strain (a CTN-1V strain) in a diploid cell MRC-5 strain. The preparation method of the rabies virus strain comprises the following steps: 1) taking MRC-5 cells in a cell bank for resuscitation, cultivating for 3 days for forming single layer cells, pouring away a cell culture fluid in an MRC-5 cell culture flask, adopting a 0.25% of trypsin digestive juice for digestion, dispersing into uniform cells, inoculating a rabies virus CTN-1V5 strain suspension according to the dosage of 0.01-1.0 MOI, cultivating at 37 DEG C for 2-3 days, replacing the suspension with a maintenance medium containing 2-4% of bovine serum, cultivating at 33-35 DEG C for 3-5 days to obtain a virus liquid, and cryopreserving at -70 DEG C to prepare a virus suspension; 2) adopting the method, and continuously subculturing for 30-32 generations on MRC-5 cells. The preservation code of the rabies vaccine virus strain (an SNK-CTN strain) is CGCC No8887.

Description

technical field [0001] The invention relates to the field of microbes and biopharmaceuticals, in particular to a rabies virus strain CTN-1V adapted to a diploid cell MRC-5 strain. 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 stromal cells used in the production of rabies vaccines in the world mainly include animal-derived cells such as primary hamster kidney cells, primary chicken embryo cells, and African green monkey kidney passage cells (Vero cells). Animal-derived primary cells cannot eliminate the contaminat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/205A61P31/14G01N33/569C12R1/93
Inventor 崔栋卜海兵赵荣辉
Owner SHINAIKE JIANGSU BIOLOGICAL PHARMA
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