DNA fragment and application thereof in construction of recombinant influenza virus expressing red fluorescent protein gene

A technology of red fluorescent protein, influenza virus, applied in the field of molecular biology

Active Publication Date: 2019-09-06
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no relevant report about inserting a fluorescent marker gene (mCherry) into the influenza non-structural protein NS1 protein to make the virus express red fluorescence

Method used

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  • DNA fragment and application thereof in construction of recombinant influenza virus expressing red fluorescent protein gene
  • DNA fragment and application thereof in construction of recombinant influenza virus expressing red fluorescent protein gene
  • DNA fragment and application thereof in construction of recombinant influenza virus expressing red fluorescent protein gene

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1, the rescue of the H9N2 recombinant virus carrying mCherry gene

[0048] 1.1 Construction of NS gene full-length infectious clone carrying mCherry gene

[0049] Extract the parent virus strain (influenza virus A / chicken / Guangdong / V / 2008 (H9N2), referred to as V K627 ), design primers to amplify the gene sequences encoding NS1 protein and NEP protein (without stop codon), and insert the mCherry fluorescent gene behind the NS1 gene, which uses PTV that can be recognized and cleaved by proteases between the gene and the NEP gene -1 2A sequence connection, see the specific construction figure 1 ; wherein primers Aar I-V-NS1-F and V-NS1-mCherry-R amplify the sequence (NS1 gene sequence) encoding NS1 protein; mCherry-2A sequence is artificially synthesized by Jinweizhi Company; I-V-2A-NEP-R amplifies the gene sequence of NEP protein (NEP gene sequence), and inserts Aar I restriction sites in the upstream and downstream respectively. Primer sequences (5'-3') ar...

Embodiment 2

[0109] Example 2: V K627 - Identification and application of mCherry recombinant virus

[0110] 2.1 Identification of recombinant virus NS full-length gene and fluorescent gene mCherry

[0111] Recombinant virus V of generations P1 to P4 were extracted with the total RNA rapid extraction kit of Feijie Biotech Co., Ltd. K627 -The total RNA of mCherry was reversed using MLV reverse transcriptase (Takara code No.2641A) to obtain cDNA. The specific reverse transcription reaction system is as follows:

[0112] RTase M-MLV(RNase H-)(200U / μL) 1μL 5×M-MLV-Buffer 12μL dNTP Mixture (10mmol) 12μL RNase Inhibitor (40U / μL) 2μL Uni12primer (10pmol) 3μL RNA template 30μL total capacity 60μL

[0113] Design NS full-length identification primers (SEQ ID NO:25 and SEQ ID NO:26) and mCherry fluorescent gene identification primers (SEQ ID NO:27 and SEQ ID NO:28), the PCR reaction system is as follows:

[0114] 2×Premix Ex-Taq ...

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Abstract

The invention discloses a DNA fragment and an application thereof in construction of a recombinant influenza virus expressing a red fluorescent protein gene. An mCherry fluorescent gene is inserted behind an NS1 gene of an H9N2 influenza virus strain, wherein the complete NS gene is formed by sequentially linking an NS1 gene, the mCHerry fluorescent gene and an NEP gene; the gene and the NEP geneare connected by a PTV-1 2A sequence which can be recognized and cut by protease, so a fluorescent protein-labeled NS fusion gene is constructed and has the nucleotide shown in SEQ ID NO:1. The constructed recombinant influenza virus can produce live virus expressing red fluorescent proteins, is relatively stable in the process of virus passage, does not affect the function of the virus itself, has the growth curve and the titer similar to those of a wild virus strain, can be used for subsequent research, and is of great significance on development, screening and production of anti-influenza virus drugs.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a DNA segment and its application in constructing recombinant influenza virus expressing red fluorescent protein gene. Background technique [0002] Influenza viruses belong to the single-stranded negative-sense RNA virus of the Orthomyxoviridae family and are divided into three types: A (A), B (B), and C (C). Among them, the natural host of type A virus is wild waterfowl, which can infect a variety of birds and mammals, and is the pathogen that causes the epidemic of human influenza and avian influenza. Since the first report of avian influenza in 1978, it has caused many pandemics around the world, leading to large-scale deaths of humans and poultry, and seriously threatening the poultry industry and public health security. Furthermore, according to recent studies, seasonal influenza affects about 20% of the world's population and causes between 250,000 and 500,000 deaths per ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N7/01C12N15/63C12N15/66
CPCC07K14/43595C12N7/00C12N2760/16021
Inventor 亓文宝周傲白雪劳光杰廖明李华楠李小康
Owner SOUTH CHINA AGRI UNIV
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