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Recombinant influenza virus and application thereof

An influenza virus and recombinant virus technology, applied in antiviral agents, virus/phage, recombinant DNA technology, etc., can solve the problems of limited VLP production, lack of mastery of VLP technology, and difficulty in development, and achieve convenient experimental detection and simple steps. , the effect of avoiding interference

Inactive Publication Date: 2015-01-28
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2009, the Sandbulte MR research team used virus-like particles (VLP) containing only influenza virus NA for the detection of anti-NA antibodies. This method has applied for a patent. Due to the limited yield of the prepared VLP, there may also be baculovirus in the system. It is difficult for laboratories or vaccine manufacturers who lack the knowledge of VLP technology to carry out

Method used

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  • Recombinant influenza virus and application thereof
  • Recombinant influenza virus and application thereof
  • Recombinant influenza virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, recombinant virus RGH6N1 (Br59NA), RGH6N2 (Br10NA) preparation and its reaction to NA antibody

[0056] 1. Rescue of recombinant viruses RGH6N1 (Br59NA) and RGH6N2 (Br10NA)

[0057] 1. Amplification of influenza virus H6 and NA genes

[0058] The HA gene has different subtypes, one of which is H6;

[0059] Viral RNA was extracted using RNeasy Mini Kit (Qiagen, cat#74104), 200 μL of virus was added to 500 μL of lysate, and 50 μL of viral RNA was amplified by one-step RT-PCR.

[0060] The H6 gene was amplified by overlapping PCR using A / Env / 2029 / 2011 (H6N1, the virus was collected by the Influenza Department of the Institute of Viral Disease Control, Chinese Center for Disease Control and Prevention) RNA as a template to obtain a PCR product.

[0061] The NA gene was amplified by one-step PCR using the RNA of different subtypes of virus strains as templates. The different subtypes of virus strains were A / Brisbane / 59 / 2007(H1N1), A / Brisbane / 10 / 2007(H3N2), A...

Embodiment 2

[0129] Example 2, Preparation of recombinant virus RGH6N9 (AH1NA) and its response to anti-N9 antibody

[0130] 1. Rescue of recombinant virus H6N9 (AH1NA)

[0131] 1. Amplification of influenza virus H6 and NA genes

[0132] 1), the acquisition of influenza virus H6 gene

[0133] Same as one, 1, 1) of Example 1, the H6 gene PCR product (sequence 1) was obtained.

[0134] 2) Acquisition of NA gene

[0135] A / AH1 / 2013 (H7N9) RNA was used as template, and INF-NA-F and INF-NA-R were used as primers for RT-PCR amplification to obtain a 1444bp NA gene (H7N9) PCR product (after sequencing, with sequence Nucleotides shown in sequence 4 in the list);

[0136] The reaction system and conditions are the same as in Example 1.

[0137] 2. Obtaining of 8 recombinant vectors for packaging recombinant viruses

[0138] Use BsmB I to linearize the PHW2000 vector and the H6 gene PCR product for Infusion connection to obtain the recombinant vector PHW2000-A / Env / 2029 / 2011_HA (after sequenci...

Embodiment 3

[0168] Example 3, Preparation of recombinant virus RG H6N1 (CA04NA) and its response to anti-NA antibodies

[0169] 1. Rescue of recombinant virus RG H6N1 (CA04NA)

[0170] 1. Amplification of influenza virus H6 and NA genes

[0171] 1), the acquisition of influenza virus H6 gene

[0172] Same as one, 1, 1) of Example 1, the H6 gene PCR product (sequence 1) was obtained.

[0173] 2) Acquisition of NA gene

[0174] Using the RNA of A / California / 04 / 2009 (H1N1) as a template, INF-NA-F and INF-NA-R as primers for RT-PCR amplification, the NA gene (2009H1N1) PCR product of 1458bp was obtained (through sequencing, have the nucleotide shown in sequence 5 in the sequence listing);

[0175] The reaction system and conditions are the same as in Example 1.

[0176] 2. Obtaining of 8 recombinant vectors for packaging recombinant viruses

[0177] Use BsmB I to linearize the PHW2000 vector and the H6 gene PCR product for Infusion connection to obtain the recombinant vector PHW2000-A / E...

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Abstract

The invention discloses a recombinant influenza virus and application thereof and provides a preparation method of the recombinant virus. The preparation method comprises the following steps: guiding an HA6 subtype influenza virus hemagglutinin encoding gene, an influenza virus neuraminidase (NA) gene and six genes including PB2, PB1, PA, NP, M and NS in an influenza virus into a host cell together, and packing to obtain the recombinant virus. The invention also discloses a detection method for detecting antibodies for inhibiting activities of seasonal influenza N1, N2, 2009H1N1N1 and 2013H7N9N9 enzymes by virtue of the recombinant virus. The detection method comprises the steps of sample treatment and result analysis. According to the invention, the immune response to NA of people can be rapidly and effectively measured, the operation is simple, the application is convenient, and viable technical support is provided for clinical detection and vaccine evaluation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant influenza virus and its application. Background technique [0002] Influenza viruses belong to the Orthomyxoviridae family and are segmented negative-sense RNA viruses. According to the nucleoprotein (NP) and matrix protein (M) of the virus, it is divided into type A, type B, and type C. Influenza A has a high degree of variability and a wide range of infected hosts, posing the greatest threat to public health. Influenza A viruses are further divided into HA1-16 subtypes according to the hemagglutinin (HA) on the surface of the virus, and NA1-9 subtypes according to neuraminidase NA. Influenza A viruses currently circulating in the population mainly include H1, H2, H3 and N1, N2 subtypes. In recent years, the new influenza viruses 2009H1N1 and H7N9 avian influenza viruses that have infected humans, the former caused influenza pandemics, and the latter's fatality rate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85G01N33/573A61K39/145A61P31/16
Inventor 周剑芳秦堃朱云白天王大燕舒跃龙
Owner 中国疾病预防控制中心病毒病预防控制所
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