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Hepatitis A virus strain SH and diploid cell adaptation method thereof

A technology of hepatitis A virus and diploid cells, applied in the field of microorganisms, can solve the problem that hepatitis A vaccine cannot meet the domestic market demand, and achieve the effect of short reproduction cycle and high reproduction efficiency

Active Publication Date: 2011-09-07
SHENZHEN KANGTAI BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country has a large population, and the hepatitis A vaccine is far from meeting the domestic market demand

Method used

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  • Hepatitis A virus strain SH and diploid cell adaptation method thereof
  • Hepatitis A virus strain SH and diploid cell adaptation method thereof
  • Hepatitis A virus strain SH and diploid cell adaptation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 The isolation method of hepatitis A virus strain SH

[0027] (a) A total of 30-50 g of feces in the acute phase of four clinical hepatitis A patients (patients admitted to the Hepatitis Department of Shenyang Sixth People's Hospital) were collected, and 40 glass beads with a diameter of 3 mm and 5 times the volume of PBS were added and shaken for 10 minutes. minute;

[0028] (b) centrifuge at 3000 rpm for 20 minutes, and collect the supernatant;

[0029] (c) Treat the precipitate twice more according to the above method, collect and combine the supernatants for three times;

[0030] (d) Add PEG6000 10g / 100ml and NaCl 2.3g / 100ml to the supernatant, and place it at 4°C for several hours after all are dissolved;

[0031] (e) Centrifuge at 8000 rpm for 30 minutes, discard the supernatant;

[0032] (f) Resuspend the precipitate in 50ml PBS, add an equal volume of chloroform and shake for 20 minutes; centrifuge at 3000 rpm for 30 minutes, collect the upper liquid...

Embodiment 2

[0036] Example 2 Adaptation of Hepatitis A Virus Strain SH on MRC-5 Cells

[0037] 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 30-37°C, culture it at 37°C, and grow into single cells after 3-4 days. Inoculate the hepatitis A virus strain SH strain suspension that embodiment 1 isolates after the layer, 40cm 2 Seed 1ml virus suspension in the culture bottle, absorb at 37°C for 2 hours, add 20ml maintenance solution (MEM plus 2% calf serum), culture at 35°C, replace the virus maintenance solution once every 7 days, cultivate for 35 days, use PBS Wash the cell surface 3 times, then digest the cells with 0.125w / v% trypsin, press 0.01ml / cm 2 Add MEM to the culture area to suspend and precipitate cells, which is the virus cell suspension, and ultrasonically disrupt the cells to prepare a virus suspension.

[0038] According to the above method, the SH strain of hepatit...

Embodiment 3

[0065] Example 3 Application of Hepatitis A Virus Strain SH in the Preparation of Vaccines and Drugs for Prevention and Treatment of Hepatitis A

[0066] Take human embryonic lung diploid cells (MRC-5) cultured in a monolayer cell factory, digest the cells with 0.125w / v% trypsin-EDTA, add MEM cell culture medium, so that the cell concentration is 100 per ml - 1.5 million cells, the cell fluid is added to the hepatitis A virus at a MOI of 0.05-0.1 and mixed and adsorbed for 30-90 minutes at a temperature of 20-30°C. Dilute the mixed and adsorbed cell-virus mixture 10 times with MEM cell culture medium containing 10% calf serum, then inoculate at 0.5 MOI in cell factories with layers 2-40 to proliferate hepatitis A virus, and culture at 35°C±0.5°C for 21 -24 days. At the peak of virus proliferation, the cells were routinely digested with 0.125w / v% trypsin, and 20 μl of 0.01M PBS with a pH value of 7.2 was added per square centimeter to collect the cell-virus mixture. The cell-...

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Abstract

The invention provides a new hepatitis A vaccine virus strain SH and a separation method as well as an MRC-5 cell adaptation method thereof. The virus is separated from excrement of a hepatitis A acute infection patient and is transferred to a diploid cell MRC-5 for adaptation culture. The virus strain SH is proved to be a hepatitis A virus through methods such as gene sequencing, neutralization test and the like; and during adaptation, early-generation sub-culturing period is 35 days, and culturing period is shortened to be 24 days after the strain is sub-cultured for 8 generations. After the strain is continuously cultured for 8 generations, antigen titer can reach (1:512)-(1:1,024), and the virus infection titer can reach 7.0 to 8.01gCCID50 / ml. Immunogenicity tests and cross protection tests show that the strain has a good immunogenicity protection effect during production of a hepatitis A inactivated vaccine, is suitable for industrially producing the hepatitis A inactivated vaccine strain on a large scale and is an ideal strain for producing the hepatitis A inactivated vaccine.

Description

technical field [0001] The invention relates to the field of microbes, in particular to a hepatitis A virus strain SH and a diploid cell adaptation method thereof. Background technique [0002] Hepatitis A, referred to as hepatitis A, is an acute infectious disease caused by hepatitis A virus that seriously endangers human health. Hepatitis A is mainly transmitted through the "fecal-oral" route, or transmission among individuals, or outbreaks of hepatitis A are caused by water or food contaminated with hepatitis A virus. After older children and adults are infected with hepatitis A, more than 70% are clinical infections, and the case fatality rate is 0.3% to 0.6%; the case fatality rate of patients over 50 years old is 1.8%. After chronic liver disease is infected with hepatitis A, the risk of acute liver failure raised. With the improvement of living conditions, the number of adults infected with hepatitis A tends to increase, and the proportion of clinical hepatitis A in...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/08A61K39/29A61P31/14A61P1/16C12R1/93
CPCC12N2770/32464A61K39/12C12N2770/32434C12N2770/32421C12N7/00C12N7/08A61K39/29A61K2039/5252A61P1/16A61P31/14
Inventor 张现臣魏文进黄秋香钟汉斌孟红彦王春雨
Owner SHENZHEN KANGTAI BIOLOGICAL PROD
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