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Reassortant influenza virus production method

An influenza virus, reassortment technology, applied in the direction of viruses, biochemical equipment and methods, pharmaceutical formulations, etc., can solve the problems of preparing target gene recombinants, etc., and achieve the effect of high proliferation

Active Publication Date: 2019-02-05
THE RES FOUND FOR MICROBIAL DISEASES OFOSAKA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the reassortment method using chicken embryos as a host, there is a problem that it is not always possible to produce a target gene recombinant

Method used

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  • Reassortant influenza virus production method
  • Reassortant influenza virus production method
  • Reassortant influenza virus production method

Examples

Experimental program
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preparation example Construction

[0062] According to the preparation method of the present invention, the target reassortant influenza virus can be obtained in less than half the time of the prior art. If the existing neutralizing antibody or antiserum can be used, the target reassortant influenza virus can be obtained preferably within 17 days, more preferably within 15 days, further preferably within 13 days, most preferably within 10 days. When new antiserum needs to be prepared, preferably the target reassortant influenza virus can be obtained within 24 days, more preferably within 20 days, further preferably within 16 days, most preferably within 12 days. The method of the present invention is very useful when early acquisition of a vaccine is required, such as during an influenza pandemic.

[0063] In this specification, the efficiency of gene recombination is used to indicate the ease of preparation of genetic recombinants of influenza virus. The gene recombination efficiency indicates the ratio of th...

example 1

[0073] (Reference Example 1) Live virus was used to prepare reassortant influenza virus

[0074] Reassortant influenza viruses were prepared using live viruses without ultraviolet irradiation (hereinafter referred to as "UV irradiation") for antigenic strains.

[0075] 1. The virus strain used and the antiserum used

[0076] First influenza virus strain (antigenic strain): A / California / 7 / 2009(H1N1)pdm09 (hereinafter referred to as "CA / 7")

[0077] Second influenza virus strain (donor strain): A / Ibaraki / N12232 / 2012 (H3N2) (hereinafter referred to as "IB / 232")

[0078] Anti-donor strain serum: the ferret infection serum of the donor strain (HI antibody titer: 1280) was subjected to RDE treatment, so that the final dilution factor was 2 times.

[0079] 2. Preparation of reassortant influenza virus

[0080] 1) Using Eagle MEM medium containing glutamine (4mM), glucose (4.6g / L), sodium bicarbonate (20mM), and 0.1×TrypLESelect (hereinafter referred to as "medium for virus culture...

example 2

[0103] (Reference Example 2) Confirm the impact of the virus amount of the antigenic strain on the preparation of reassortant influenza virus

[0104] In this reference example, the effect of the virus amount of the antigenic strain on the gene recombination efficiency was studied. In addition, the virus used and the antiserum used were the same as those in Reference Example 1.

[0105] 1. Preparation of reassortant influenza virus

[0106] 1) Use medium for virus culture to prepare 10 7 TCID 50 / mL donor strain solution, at the same time, prepared 10 2 TCID 50 / mL, 10 7 TCID 50 / mL of the antigenic strain solution.

[0107] 2) at 25cm 2 MDCK cells (the MDCK cells identified by the international deposit number NITE BP-02014) were cultured to confluence (about 5×10 6 cells / flask), then remove the medium, inoculate 200 μL of the antigenic strain solution, and incubate at 34°C, 5% CO 2 Incubated for 1 hour. Then, 200 µL of the donor strain solution was inoculated, and ...

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Abstract

Provided is a method for producing a reassortant influenza virus having genome segments from two or more influenza virus strains. This method for producing a reassortant influenza virus containing anantigen protein from a first influenza virus strain comprises the following steps: 1) A step in which the first influenza virus strain is irradiated with ultraviolet rays at a dose at which the virushas reduced or no proliferation capacity and maintains initial infectivity; 2) a step in which a host is infected with the first influenza virus strain and a second influenza virus strain; 3) a step in which the host infected with the first influenza virus strain and the second influenza virus strain is cultured to obtain a culture; 4) a step in which, from the culture obtained at step 3), an influenza virus strain having an antigen protein of the second influenza virus strain is deactivated; and 5) a step in which a reassortant influenza virus is recovered following step 4).

Description

technical field [0001] The invention relates to a preparation method of a reassortant influenza virus. [0002] This application claims the priority of Japanese Application No. Japanese Patent Application No. 2016-140366, which is incorporated herein by reference. Background technique [0003] Influenza is an infectious disease that spreads around the world every year and is caused by influenza virus. Influenza viruses belong to the Orthomyxoviridae family and have an envelope with a lipid bilayer membrane structure. Influenza viruses are divided into three types: A, B, and C, which are called influenza A, influenza B, and influenza C, respectively. In general, influenza virus refers to type A or type B in particular. The difference between type A, type B, and type C is based on the difference in antigenicity of M1 protein (matrix protein) and NP protein (nucleoprotein) among the proteins constituting the virion. In addition, even if the same type is A or B, the molecule...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02A61K39/00A61K39/145
CPCA61K39/00A61K39/145C12N7/00C12N2760/16121C12N2760/16151C12N2760/16021C12N2760/16051C12N13/00A61K2039/525
Inventor 藤本贵男藤田顺二
Owner THE RES FOUND FOR MICROBIAL DISEASES OFOSAKA UNIV
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