Enhanced green fluorescent protein recombinant H5N1 subtype influenza virus and its application

A green fluorescent protein, influenza virus technology, applied in recombinant DNA technology, virus/phage, introduction of foreign genetic material using vectors, etc., can solve the problem that there is no recombinant H5N1 subtype influenza virus.

Active Publication Date: 2014-02-05
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no report on the recombinant H5N1 subtype influenza virus with enhanced green fluorescent protein (EGFP) as the reporter gene

Method used

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  • Enhanced green fluorescent protein recombinant H5N1 subtype influenza virus and its application
  • Enhanced green fluorescent protein recombinant H5N1 subtype influenza virus and its application
  • Enhanced green fluorescent protein recombinant H5N1 subtype influenza virus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Obtaining and Identification of Recombinant H5N1 Subtype Influenza Virus

[0062] 1. Construction of recombinant expression vector pHW-VN-NA-EGFP

[0063] 1. Primer design

[0064] According to the EGFP gene sequence in the pEGFP-C1 plasmid and the NA segment sequence in the VN1194 virus genome (RNA fragment 6 encoding the NA protein, whose sequence is shown in sequence 6 in the sequence table), it is designed to amplify the NA gene (including 3 '- non-coding region), EGFP gene and 5'-packaging signal primers, and introduce porcine teschovirus-1, PTV-1 2A peptide sequence (the first sequence of sequence 9) between the EGFP gene and the NA gene 1368-1433 positions), the restriction endonuclease BsaⅠ was added to the end of the forward primer (PF-NA) used to amplify the NA gene and the reverse primer (PR-NA) used to amplify the 5'-packaging signal The recognition site (underlined italic part), the specific primer sequence and the length of the amplified produ...

Embodiment 2

[0103] Example 2. Screening of Anti-H5N1 Subtype Influenza Virus Drugs Using Recombinant Virus rVN-EGFP

[0104] In this example, the recombinant virus rVN-EGFP constructed in Example 1 is used to detect whether the drug to be tested has anti-H5N1 subtype influenza virus activity. Among them, the existing anti-influenza drug oseltamivir phosphate (Tamiflu) was used as the antiviral drug to be tested, thereby verifying that the recombinant virus rVN-EGFP constructed in Example 1 can be used for anti-H5N1 subtype influenza virus drugs filter. The specific operation is as follows:

[0105] (1) Transfer MDCK cells to a 24-well cell plate, count the cells after the cells are full the next day, and then dilute the recombinant virus rVN-EGFP constructed in Example 1 with DMEM medium according to the cell count results, and make 100 μL of After the virus dilution solution infects the cells, it reaches 1 PFU / cell or 0.1 PFU / cell, that is, the cells are infected with the virus amount ...

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Abstract

The invention discloses an enhanced green fluorescent protein recombinant H5N1 subtype influenza virus and its application. The genome of the recombinant H5N1 subtype influenza virus is composed of eight RNA fragments of the following a) and b): a) seven RNA fragments which are same as the genome of wild type H5N1 subtype influenza virus contains RNA fragment 1 encoding PB2 protein, RNA fragment 2 encoding PB1 and PB1-F2 proteins, RNA fragment 3 encoding PA protein, RNA fragment 4 encoding HA protein, RNA fragment 5 encoding NP protein, RNA fragment 7 encoding M1 and M2 proteins, RNA fragment 8 encoding NS1 and NS2 proteins; b) fragment 6' which is obtained by the insertion of RNA fragment containing green fluorescent protein coding RNA to 5'-noncoding region of RNA fragment 6 encoding NA protein in the genome of wild type H5N1 subtype influenza virus. According to the invention, rapid quantitative determination of the recombinant virus can be performed by observation of fluorescence positive cell number and fluorescence intensity, cumbersome procedure of regular titration is saved, and a convenient and effective tool for anti-H5N1 subtype influenza virus drug screening and vaccine evaluation is provided.

Description

technical field [0001] The present invention relates to a recombinant H5N1 subtype influenza virus and application thereof, in particular to a recombinant H5N1 subtype influenza virus using enhanced green fluorescent protein (EGFP) as a reporter gene and application thereof. Background technique [0002] The H5N1 subtype influenza virus is a type of influenza A virus that is mainly transmitted in birds. In recent years, reports of H5N1 subtype influenza virus infecting humans have increased year by year. As of August 2012, 608 people have been reported infected in 15 countries and regions around the world, of which 359 people died, with a mortality rate as high as 59%. Infection titer is the basic data required for virus-related basic research and drug and vaccine development. At present, the infectious titer of H5N1 subtype influenza virus is mainly determined by measuring half the tissue culture infectious dose (TCID50) or plaque forming unit (PFU) of the virus, the opera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/85C12N5/10C12N1/21C12Q1/70C12Q1/02C12R1/93
Inventor 李靖户义杨银辉祝庆余秦成峰孙伟康晓平吴晓燕韩鹏飞张雨李裕昌李佳明
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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