Influenza B virus Vero cell productive adaptive strain and preparation and application thereof

A type of influenza B virus and influenza B technology, applied in antiviral agents, viruses/bacteriophages, biochemical equipment and methods, etc., can solve problems such as complex processes, difficulty in large-scale production, and contamination by external factors, and achieve production The process is easy, the yield is improved, and the effect of avoiding the easy change of the hemagglutinin antigen

Active Publication Date: 2010-01-06
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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  • Influenza B virus Vero cell productive adaptive strain and preparation and application thereof
  • Influenza B virus Vero cell productive adaptive strain and preparation and application thereof
  • Influenza B virus Vero cell productive adaptive strain and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation method of the high-yield adapted strain of influenza B virus Vero cells with the preservation number CGMCC No.2931:

[0053] 1) Inoculate the influenza B virus isolated from clinical samples onto Vero cells that have grown into a dense monolayer, use DMEM / F12 as the basal medium, and add TPCK-trypsin to a concentration of 1ug / ml, bovine Serum albumin 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;

[0054] 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 high-yield adaptation strain of influenza virus Vero cells, the hemagglutination titer is maintaine...

Embodiment 2

[0057] Influenza virus B / Yunnan / 2 / 2005va (B) maintained a stable and high yield in continuous passage on serum-free cultured Vero cells.

[0058] 1) The influenza virus Vero cell adaptation strain B / Yunnan / 2 / 2005va (B) gained in embodiment 1 is inoculated on the Vero cells that have grown into a dense monolayer in 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;

[0059] 2) re-inoculate the virus liquid harvested from the first generation obtained in the above 1) step on the Vero cells cultured without serum, and after culturing with the same method and conditions as 1), harvest the virus liquid; The virus was subcultured, and the hemagglutination titer was measured after repeated freezing and thawin...

Embodiment 3

[0061] Using the influenza virus Vero cell high-yielding adaptive strain B / Yunnan / 2 / 2005va (B) obtained in Example 1 as the donor strain, and reassorting with B / xinjian / 1 / 2006 to obtain the Vero of the influenza B virus Victoria lineage strain Methods for cell-adapted strains:

[0062] 1) Influenza virus reassortment

[0063] Mix Vero cell non-adapted strain B / xinjian / 1 / 2006 (Victoria lineage) and influenza virus Vero cell-adapted strain B / Yunnan / 2 / 2005va (B) at a volume ratio of 9:1, and inoculate 9-10-day-old SPF chickens Embryos, inoculate 0.2ml per embryo, incubate at 33±1°C for 24 hours, cool the embryos overnight at 2°C, and collect the allantoic fluid of chicken embryos as the virus harvesting liquid;

[0064] 2) Breeding of Vero cell-adapted strain of influenza virus B / xinjian / 1 / 2006 (Victoria lineage) after reassortment

[0065] Mix the virus liquid harvested in step 1 with the anti-B / Yunnan / 2 / 2005va (B) antiserum with an antibody hemagglutination inhibition titer of ...

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Abstract

The invention provides an influenza B virus Vero cell productive adaptive strain and a preparation method and application thereof. The influenza B virus Vero cell productive adaptive strain is named as B/Yunnan/2/2005va(B) with a preserved number of CGMCC No.2931. The virus strain can be continuously subcultured on the Vero cell, and a clotting titer can be kept over 1: 512. Through detecting, the virus strain is the influenza B virus, belonging to a Yamagata pedigree. The virus strain includes eight genetic fragments of the influenza B virus and has the characteristic of continuously producing the Vero cells. On the one hand, the virus strain is inoculated in the Vero cell, the supernate of a culture is collected to obtain a influenza B virus vaccine which has favorable immunogenicity and proactive effects through an experiment; on the other hand, the virus strain also can be used as a donor virus strain which is reassorted through a natural selective pressure or a reverse genetics to obtain the adaptive strain of an epidemic strain on the Vero cell, thereby an epidemic strain Vero cell influenza vaccine is prepared.

Description

technical field [0001] The invention relates to a high-yield adaptive strain of influenza B virus with sustained high-yield on Vero cells, and also relates to a vaccine prepared therefrom and a preparation method of a reassorted Vero cell-adapted strain, which belongs to the technical field of biological products. Background technique [0002] Influenza B (flu) virus is one of the main pathogens of influenza. Influenza B virus has only one subtype and has strong host specificity. So far, it has only been found to infect humans and seals. Like influenza A virus, it also depends on the mutation of the virus outer membrane protein, especially the heavy chain region (HA1) of hemagglutinin (hemagglutinin), to change the antigenic characteristics to evade the existing immune protection of the body, thus continuously leading to the pathogenesis of influenza. Popularity. Therefore, the influenza B virus antigen is an important part of the trivalent influenza vaccine. It has been ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/08A61K39/145A61P31/16C12N15/85C12N7/01C12R1/93
Inventor 廖国阳刘泽孙明波李卫东高菁霞马磊周健张新文蔡玮姜述德
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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