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Hepatitis A virus strain HAV-ZL2012, vaccine prepared by using same and application thereof

A technology of hepatitis A virus and virus strains, applied in the field of bioengineering, can solve the problems of long growth cycle and high production cost, and achieve the effects of weakened pathogenicity, shortened replication cycle, and enhanced virus replication ability

Active Publication Date: 2014-01-29
内蒙古必威安泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The establishment and identification of hepatitis A virus vaccine strains have contributed to the widespread application of hepatitis A vaccines worldwide, but the current hepatitis A vaccines have the characteristics of high production costs, long growth cycle of hepatitis A virus, and no cytopathic changes on host cells. Make the production and quality control cycle of vaccines as long as half a year

Method used

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  • Hepatitis A virus strain HAV-ZL2012, vaccine prepared by using same and application thereof
  • Hepatitis A virus strain HAV-ZL2012, vaccine prepared by using same and application thereof
  • Hepatitis A virus strain HAV-ZL2012, vaccine prepared by using same and application thereof

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Embodiment 1

[0034] The isolation and identification of embodiment 1 hepatitis A virus strain

[0035] The feces of patients in the acute stage of Yanshan County Hospital, Hebei, were dissolved in PBS (pH7.2-7.4), made into a 30-50% suspension, centrifuged, ultracentrifuged with 30% sucrose, and then washed with CsCl 2 Gradient centrifugation, the density gradient is between 1.0 and 1.6g / ml, measure the hemagglutination titer, the hemagglutination titer is 1:32, and further dilute it with 1:5 cell culture medium containing 1.0% fetal bovine serum, and use Filter through a 0.45ml pinhole filter, then sterilize and filter through a 0.22μm pinhole filter, inoculate a monolayer of FRhK6 cells, place the inoculum at 37°C for 4 hours, remove the liquid, and replace it with 0.5% fetal bovine serum, 100IU Qing Damycin, 10 μg streptomycin / ml MEM culture solution, replace the culture solution every week, culture at 37°C for 60 days, use ELISA-TCID 50 Methods The hepatitis A virus antigen was sequen...

Embodiment 2

[0036] Subculture of embodiment 2 hepatitis A virus strain

[0037] 1. After the positive virus strain in Example 1 is passed on the monolayer cells of FRhK6 for 10 times, it is transferred to Vero cells, and the Vero cells are passed to the 10th generation, and the virus culture solution is passed through ELISA-TCID 50 , using the 2B-2C interval of the virus strain genome as a template to design primers for RT-PCR detection, the result was positive, and the virus titer reached 10 4 logELISA-TCID 50 / ml;

[0038] 2. Attenuated adaptation on diploid cells

[0039] The cells obtained in step 1 uploaded to the 10th generation of Vero cells were inoculated with monolayer diploid MRC-5 cells at an MOI of 0.01-0.1, and passed to the 5th-8th generation, and the virus titer could reach 10 7 logELISA-TCID 50 / ml, using the 2B-2C interval of the virus strain genome as a template to design primers for RT-PCR detection, the result is positive, and the diploid cell-adapted strain of th...

Embodiment 3

[0048] Embodiment 3 inactivated vaccine preparation

[0049] Using a bioreactor, Vero cells were cultured in suspension with a cell density of 1-2×10 8 ml, the attenuated and adapted hepatitis A virus strain HAV-ZL2012 of the present invention was inoculated at a MOI of 0.01, and the virus was continuously cultured and digested by perfusion for two weeks at 37°C. , 500K medium volume fiber column concentrated SephawsebFF or Eephorose4FF purification or sucrose density continuous flow ultracentrifugation purification, adding formalin solution at 1:4000 (v / v) to inactivate at 37°C for 3 days, residual formalin was washed with double Sodium thiosulfate neutralization, all operations must be carried out under sterile conditions.

[0050] The inactivated vaccine antigen 1m is one dose, and 4 doses are injected into rhesus monkeys intravenously to generate hepatitis A antibody, or 4 doses are injected into rhesus monkeys to challenge the virus.

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Abstract

The invention discloses a hepatitis A virus strain HAV-ZL2012, a vaccine prepared by using same and application thereof. According to the invention, through the adoption of a reverse molecular biology technology and a novel subculturing method, the adaption and attenuation of a virus strain obtained through primary isolation are realized and rapidly facilitated; and growth, immunity and safety studies show that the virus strain HAV-ZL2012 can be used for preparing both live attenuated vaccines and inactivated vaccines. According to the invention, through the adoption of a rapid attenuation and adaption method of the hepatitis A virus, the growth period of the hepatitis A virus is reduced from 100 days to 14 days. The generation order of subculture adaption on a diploid cell is reduced from 40 generations to 10 generations; the generation order of subculture adaption on a subculture cell is reduced to 10-15 generations; the production period and cost are greatly reduced; and a great promoting effect on the development of a hepatitis A vaccine is obtained.

Description

technical field [0001] The present invention relates to a hepatitis A virus strain, a vaccine prepared from the virus strain and its application, in particular to a hepatitis A virus strain HAV-ZL2012, an attenuated live vaccine and an inactivated vaccine prepared therefrom. The invention also provides an attenuation adaptation method of the virus strain. Belongs to the field of bioengineering. Background technique [0002] Viral hepatitis A, referred to as hepatitis A and hepatitis A, is caused by hepatitis A virus (Hapatitis Avirus, HAV). , Loss of appetite, hepatomegaly, and abnormal liver function are the main manifestations. In some cases, jaundice occurs. The main manifestation is acute hepatitis, which is common in asymptomatic infections. The disease can occur at any age, but mainly affects children and adolescents. The clinical symptoms of hepatitis A in adults are generally more severe than those in children. Winter and spring are often the peak period of hepat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/08A61K39/29A61P31/14A61P1/16G01N33/576C12R1/93
CPCY02A50/30
Inventor 吕宏亮张澍
Owner 内蒙古必威安泰生物科技有限公司
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