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Construction method of Hela cell strain capable of stably expressing H1N1 influenza virus RNPs

An influenza virus, stable expression technology, applied in the field of biomedicine, can solve problems such as non-existence

Inactive Publication Date: 2017-02-22
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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Problems solved by technology

[0004] The object of the present invention is to provide a method for constructing a Hela cell strain stably expressing H1N1 influenza virus RNPs, aiming to solve the problem that the prior art does not exist Hela-RNP cells that use the RdRp trimer interaction region as a target to study antiviral drugs Strain problem

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  • Construction method of Hela cell strain capable of stably expressing H1N1 influenza virus RNPs
  • Construction method of Hela cell strain capable of stably expressing H1N1 influenza virus RNPs
  • Construction method of Hela cell strain capable of stably expressing H1N1 influenza virus RNPs

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[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0017] The embodiment of the present invention provides a method for constructing a Hela cell strain stably expressing H1N1 influenza virus RNPs, comprising the following steps:

[0018] S01. Design primers for the amplification of H1N1 influenza virus ribonucleoprotein RNPs, PCR amplify the target fragments PB1, PB2, PA and NP, and construct recombinant plasmids pcDNA3.1 / Zeo-PB1, pcDNA3.1 / Hygro-PB2, pcDNA3. 1 / Neo-PA, pEF6 / V5-HisA-NP;

[0019] S02. Provide Hela cells, use four kinds of antibiotics Zeocin, Hygromycin B, Geneticin, Blasticidin S HCl to infect the He...

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Abstract

The invention belongs to the field of physical medicine and provides a construction method of a Hela cell strain capable of stably expressing the H1N1 influenza virus RNPs. The method comprises the steps of constructing a recombinant plasmid pcDNA 3.1 / Zeo-PB1, pcDNA 3.1U / Hygro-PB2, pcDNA 3.1 / Neo-PA, and pEF 6 / V5-HisA-NP; acquiring an optimal screening concentration for four antibiotics Zeocin, Hygromycin B, Geneticin, Blasticidin S HCl-infected Hela cells; transfecting the Hela cell strain to the recombinant plasmid pcDNA 3.1 / Zeo-PB1, the pcDNA 3.1U / Hygro-PB2, the pcDNA 3.1 / Neo-PA, and the pEF 6 / V5-HisA-NP in batches, and screening to obtain the cell strain Hela-RNP.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for constructing a Hela cell strain stably expressing H1N1 influenza virus RNPs. Background technique [0002] Influenza viruses belong to the Orthomyxoviridae family of single-stranded, negative-strand RNA enveloped viruses, which can be divided into three types: A (A), B (B), and C (C). There are many subtypes of influenza viruses and they are constantly mutating, which makes the classic anti-influenza drugs prone to drug resistance and cross-resistance. For example, the resistance of H7N9 influenza virus to amantadine in 2013 is caused by the amino acid residues of the virus M2 protein. S31N mutation; and there are few effective drugs for severe influenza patients. At present, anti-influenza virus drugs on the market mainly include M2 ​​ion channel blockers and viral neuraminic acid inhibitors (NAI). However, with the continuous evolution of influenza viruses,...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
Inventor 房师松王昕丁小满张仁利
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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