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Influenza A virus Vero cell adapted strain and application thereof

An influenza virus and adapted strain technology, applied in the directions of antiviral agents, viruses/phages, inactivation/attenuation, etc., can solve the problems of complex process, difficulty in large-scale production, contamination by exogenous factors, etc., to avoid the reduction of efficacy. Effect

Active Publication Date: 2009-10-21
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, chicken embryos are used as the substrate for preparing influenza vaccines, but there are many shortcomings, such as difficulty in large-scale production, complicated process, and the possibility of contamination by exogenous factors.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Preparation method of influenza virus Vero cell-adapted strain with preservation number CCTCC NO: V200514:

[0038] 1) Inoculate the influenza virus isolated from clinical samples onto Vero cells that have grown into a dense monolayer, use DMEM / F12 as the basal medium, add TPCK-trypsin to a concentration of 1ug / ml, bovine serum white protein to a concentration of 1ug / ml, with NaHCO 3 Adjust the pH value of the medium to 7.5; after culturing at 33±1°C for 72 hours, harvest the virus liquid and complete the first subculture;

[0039] 2) Inoculate the virus liquid harvested in the first subculture onto Vero cells again, and after culturing with the same method and conditions as step 1), harvest the virus liquid; in this way, the Vero cells are continuously passaged, and when the passage reaches 25 generations Harvest the virus liquid to obtain the Vero cell-adapted strain of influenza virus, named A / Yunnan / 1 / 2005Va (H3N2), and the preservation number is CCTCC NO: V200514....

Embodiment 2

[0041] Influenza virus Vero cell-adapted strain A / Yunnan / 1 / 2005Va (H3N2) maintains stable and high yield in continuous subculture on serum-free Vero cells.

[0042] 1) The influenza virus Vero cell adaptation strain A / Yunnan / 1 / 2005Va (H3N2) gained in Example 1 is inoculated on the Vero cells that have grown into a dense monolayer of serum-free culture, and the inoculum size is 0.2ml per small square bottle , to maintain the mother liquor composition: SFM (Hyclone), TPCK-trypsin 0.5ug / ml, bovine serum albumin 1.5ug / ml, use NaHCO 3 Adjust the pH value to 7.2, culture at 33±1°C for 72 hours, harvest the virus liquid, and complete the first generation culture;

[0043] 2) re-inoculate the virus liquid harvested from the first generation obtained in the above 1) on the Vero cells cultured without serum, and after culturing with the same method and conditions as in 1), harvest the virus liquid; The virus was subcultured, and the hemagglutination titer was measured after repeated fr...

Embodiment 3

[0045] The method for reassorting the influenza virus Vero cell-adapted strain A / Yunnan / 1 / 2005Va (H3N2) obtained in Example 1 with A / Caledonia / 20 / 99 (H1N1) to obtain the H1N1 influenza virus Vero cell-adapted strain:

[0046] ①Influenza virus reassortment

[0047] Mix Vero cell non-adapted strain A / Caledonia / 20 / 99(H1N1) and influenza virus Vero cell-adapted strain A / Yunnan / 1 / 2005Va(H3N2) at a volume ratio of 9:1, and inoculate 9-10-day-old SPF chicken embryos , inoculate 0.2ml per embryo, incubate at 33±2°C for 24h, cool the embryo overnight at 2°C, and collect the allantoic fluid of chicken embryos as the virus harvesting liquid;

[0048] ② Breeding of influenza virus H1N1 Vero cell-adapted strain after reassortment

[0049] Mix the harvested virus liquid with the anti-A / Yunnan / 1 / 2005Va (H3N2) antiserum with an antibody hemagglutination inhibitory titer of 1:640 at a volume ratio of 2:1, neutralize at 37°C for 2 hours, and inoculate Cultivate on Vero cells for 48 hours, har...

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Abstract

The invention discloses an influenza A virus Vero cell adapted strain and application thereof. The influenza A virus Vero cell adapted strain is named as A / Yunnan / 1 / 2005Va(H3N2), and the preserving number of the strain is CCTCC NO:V200514. The strain comprises an H3 subgroup hemagglutinin gene, an N2 subgroup neuraminidase gene and 6 internal genes for high yield of characteristic influenza viruses on Vero cells. The blood coagulation titer can be maintained at more than 1,024 by continuous subculturing of the strain on the Vero cells. The strain can be taken as a donor strain and subjected to genetic reassortment with an epidemic strain, or reverse genetics technology is adopted to perform genetic reassortment on the 6 internal genes and 2 surface protein genes of the epidemic strain to obtain the Vero cell adapted strain of the epidemic strain, and finally the adapted strain is used for preparing a Vero cell influenza vaccine or a Vero cell pandemic influenza vaccine.

Description

technical field [0001] The invention relates to a Vero cell-adapted strain of influenza A virus, and also relates to the reassortment of the adapted strain as a donor strain and the epidemic strain to obtain the Vero cell-adapted strain of the epidemic strain to prepare a Vero cell influenza vaccine, which belongs to the field of biotechnology . Background technique [0002] Influenza (influenza) is an acute respiratory infection caused by influenza virus. It is highly contagious and mainly spreads through air droplets. Therefore, it can occur suddenly in a short period of time and spread rapidly, causing different degrees of epidemics, even a global pandemic. , with high morbidity and certain mortality, is a worldwide infectious disease that humans cannot effectively control at present. [0003] Influenza viruses belong to the Orthomyxoviridae family. According to the antigenicity of viral nucleoprotein (NP) and matrix protein (MP), influenza viruses can be divided into th...

Claims

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Application Information

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IPC IPC(8): C12N7/08C12N15/85C12N7/01A61K39/145A61P31/16C12R1/93
Inventor 廖国阳范东瀛孙明波李卫东周健张英伟姜述德
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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