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A549-5Ps cell line and building method and application thereof

A technology of cell line and RNA polymerase, which is applied in the field of genetic engineering, can solve the problems of low sensitivity, limited vaccine protection, strong variability, etc., and achieve the effect of high sensitivity

Inactive Publication Date: 2015-04-29
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, the reporter gene used in this cell line is the puromycin resistance gene, which has problems such as long phenotype display period and low sensitivity
At the same time, the 293 cell line is not a natural target cell for influenza virus infection, which raises questions about whether the experimental results can truly reflect the natural physiological process.
[0004] Antigenic drift and variation severely limit vaccine protection in influenza A virus control
The currently approved anti-influenza drugs M2 ion channel blockers and neuraminidase inhibitors target viral targets with strong variability, resulting in rapid drug resistance of the virus

Method used

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  • A549-5Ps cell line and building method and application thereof
  • A549-5Ps cell line and building method and application thereof
  • A549-5Ps cell line and building method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] The construction of embodiment 1 expression vector

[0032] The ribosome intervention site and the puromycin resistance gene element (IRES / puro) were inserted into the pWPXLd vector to construct the pWPXLd-puro vector. Using plasmids pHW181-PB2, pHW182-PB1, pHW183-PA and pHW185-NP as templates, genes encoding influenza PB2, PB1, PA and NP proteins were respectively cloned into pWPXLd-puro vectors to construct protein expression plasmids pWPXLd-PB2, pWPXLd-PB1, pWPXLd-PA and pWPXLd-NP, the plasmid map is as follows Figure 1-5 shown.

[0033] The IRES-blasticidin sequence was inserted into the pHH21-Gluc plasmid to construct the pHH21-Gluc-IRES-blasticidin plasmid. Using the pHH21-Gluc-IRES-blasticidin plasmid as a template, the Gluc-IRES-blasticidin fragment was amplified by PCR and connected to the vector pWPXLd-puro to construct a negative-strand RNA production plasmid for the Gaussia luciferase / blasticidin resistance reporter gene pWPXLd-Gluc-IRES-blasticidin, pla...

Embodiment 2

[0035] Example 2 Cell Culture

[0036] A549 and A549-5Ps cells were cultured in DMEM medium containing 10% fetal bovine serum. 293FT cells were cultured in DMEM medium supplemented with 2mM glutamine, 0.1mM MEM non-essential amino acids and 10% fetal bovine serum.

Embodiment 3

[0037] Example 3 lentiviral production and transduction

[0038] 1×10 per well of 6-well plate 6 293FT cells. 24 hours after plating, the lentivirus packaging plasmids pMD2.G1ug, psPAX21ug and core plasmid pWPXLd-gene2ug were co-transfected, the culture medium was changed 6 hours after transfection, and the lentiviruses were harvested 48 hours after transfection to complete the lentivirus production process. The harvested lentivirus was filtered through a 0.45um sterile filter and stored at -80°C for later use.

[0039] 2×10 per well of 6-well plate 5 For A549 cells, polybrene was added to the culture medium at a final concentration of 8ug / mL. 24 hours after plating, the above virus solution was used to infect A549 cells with 1 mL of virus solution per well, and the culture medium was changed after 8 hours of infection to complete lentivirus-mediated transduction.

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an A549-5Ps cell line for stable coexpression of influenza virus A RNA polymerases PB2, PB1, PA subunit and NP protein, and resistance reporter genes of Gaussia luciferase / blasticidin, and a building method and an application of the A549-5Ps cell line. The cell line has the characteristic of monitoring replication and expression of influenza virus A genome RNA in cells in real time; and a simple and sensitive method is provided for further revelation of the transcription and replication mechanism of the influenza virus A in the cells, illumination of the interaction between viruses and hosts, and research and development of anti-influenza virus A medicines.

Description

technical field [0001] The present invention relates to genetic engineering technology, in particular, to a cell line stably co-expressing influenza A virus RNA polymerase PB2, PB1, PA subunit and NP protein and Gaussia luciferase / blasticidin resistance reporter gene and Its construction method and application. Background technique [0002] Influenza virus is the most important pathogen that causes acute respiratory infectious diseases in humans. It is mainly transmitted through air droplets. It has the characteristics of fast transmission, high incidence and often serious complications. Influenza is prevalent every year in autumn and winter in temperate regions, causing quite high morbidity and mortality, and seriously threatening human health. In 2009, the swine-origin H1N1 flu spread rapidly to 214 countries and regions around the world after the first case in Mexico, with 126,000 confirmed cases and 775 deaths in China alone. The H7N9 avian influenza virus that appeare...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/63C12Q1/02C12R1/91
Inventor 岑山王臻张永欣高茜刘振龙李晓宇
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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