Influenza virus attenuating method, influenza attenuated virus strain and application

A technology of influenza virus, virus strain, applied in the field of biomedicine

Pending Publication Date: 2022-04-22
ZHEJIAN DIFFERENCE BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently only two live attenuated vaccines on the market, and both are based on cold-adapted strains, and the use of these vaccines is limited to people aged 2 to 49 years

Method used

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  • Influenza virus attenuating method, influenza attenuated virus strain and application
  • Influenza virus attenuating method, influenza attenuated virus strain and application
  • Influenza virus attenuating method, influenza attenuated virus strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Construction of Random Base Deletion Plasmid of Influenza Virus M2 Gene

[0057] Extract the plasmid expressing the M2 gene of the influenza virus PR8 strain (the present invention has no special limitation on the construction method of this plasmid, adopt the commonly used plasmid and M gene known to those skilled in the art to carry out the construction of the plasmid overexpressing M2 according to the conventional recombinant plasmid construction method Just), using a series of base deletion primers (see Table 1 for the primer sequence) to carry out PCR amplification. After the correct molecular weight was identified by agarose gel electrophoresis, the target band was recovered and recombined at 50°C for 15 minutes, and the recombinant product was transformed into Escherichia coli competent cells. The extraction kit successfully prepared deletion plasmids PR8-M2-del14, PR8-M2-del20, PR8-M2-del22, PR8-M2-del38, PR8-M2-del65, PR8-M2-del103 (the extracted plasmid PR8- ...

Embodiment 2

[0061] Rescue and Validation of Replication-Restricted Influenza Viruses

[0062] HEK293T cells were plated in Thermo Fisher’s special six-well plate, and 12 hours later, plasmids containing 7 genes of PR8 (pFlu-PR8-PB2, pFlu-PR8-PB1, pFlu-PR8-PA, pFlu-PR8- NP, pFlu-PR8-NS, pFlu-PR8-HA, pFlu-PR8-NA), the M2 gene random base deletion series plasmids constructed in Example 1, and the plasmids expressing M2 protein were co-transfected into HEK293T cells. Change the medium 6-8 hours after transfection, freeze and thaw the cell plate once 48 hours after transfection, collect the supernatant and inoculate it into the T25 cell bottle of MDCK cells expressing M2 protein, observe the cell pathology after 72-96 hours of inoculation, and freeze the cell bottle After thawing once, the supernatant was collected by centrifugation and named rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, rPR8-M2-del103.

[0063] Utilize viral RNA extraction kit to extract the vira...

Embodiment 3

[0067] Replication-restricted influenza virus titer determination

[0068] Spread the MDCK cells expressing the M2 protein on a 96-well plate. After the cells have grown to a monolayer, take 50 μL of replication-limited influenza virus and add it to 450 μL of 1% TPCK opi-MEM, shake and mix well as system 1, and the final concentration is 10 -1 ; Take 50 μL from system 1 and add it to 450 μL 1% TPCK opi-MEM, shake and mix well as system 2, the final concentration is 10 -2 ; By analogy, the virus solution was diluted 10 times in a row and diluted to 10 times. -10 , then discard the medium in the 96-well plate, wash the plate with PBS, add 100 μL of the corresponding dilution of the virus solution to each well, do 3 repetitions for each gradient, and store at 37 ° C, 5% CO 2 After culturing for 72 hours in the cell incubator to observe the cell pathological changes, apply the Reed-Muench method to calculate its TCID 50 , virus titers are shown in Table 3.

[0069] Table 3 Sum...

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Abstract

The invention relates to an influenza virus attenuating method, an influenza attenuated virus strain and application, and belongs to the technical field of biological medicine. The attenuating method comprises the following steps: carrying out random number and position base deletion on a transmembrane structural domain and a cytoplasm structural domain of M2 protein in an influenza virus conserved region, so as to obtain the influenza attenuated virus with corresponding base deletion. The influenza attenuated virus strain obtained by the attenuating method disclosed by the invention has a good growth characteristic on an MDCK cell line for expressing M2 protein; a high dose of virus strain can grow in MDCK cells or chick embryos and has a high chicken red blood cell agglutination price; in addition, a Balb / C mouse is immunized through nasal dripping, and it is found that the virus strain is non-pathogenic to the mouse relative to the parent virus IAV PR8. The virulence of the influenza virus is reduced in a random base deletion mode, and a foundation is laid for screening of safer and more effective IAV attenuated live vaccines.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for attenuating influenza virus, attenuated influenza virus strain and application. Background technique [0002] Influenza A (IA) is a highly contagious acute respiratory disease caused by Influenza Aviruses (IAV), a segmented negative-sense RNA virus belonging to the Orthomyxoviridae family. Vaccination is the main means of preventing IA infection, and currently there are inactivated and attenuated live vaccines. Due to limited mucosal immunity and cytotoxic T cell responses, the protective effect of inactivated vaccines is only short-lived and requires annual vaccination. In contrast, intranasal immunization with live attenuated IAV vaccines elicits robust mucosal and cellular immunity, so their protective effects last longer. There are currently only two live attenuated vaccines on the market, and both are based on cold-adapted strains, and the use of these vac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N7/04C12N15/44C12N15/85C12N15/11A61K39/145A61P31/16C12R1/93
CPCC12N7/00C07K14/005C12N15/85A61K39/12A61P31/16C12N2760/16122C12N2760/16134C12N2760/16162C12N2800/107A61K2039/5254
Inventor 孙慧敏余飞周孟云毛水花方晨捷宋家升
Owner ZHEJIAN DIFFERENCE BIOLOGICAL TECH CO LTD
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