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Reporter gene cell line and construction method and application thereof

A reporter gene and construction method technology, applied in the fields of application, animal cells, genetic engineering, etc., can solve the problems that cannot be used to identify the activation state of the IFN pathway, and achieve high sensitivity results

Inactive Publication Date: 2020-08-14
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this system cannot be used to identify the state of IFN pathway activation

Method used

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  • Reporter gene cell line and construction method and application thereof
  • Reporter gene cell line and construction method and application thereof
  • Reporter gene cell line and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of recombinant expression vector pHAGE-IFN-β-Gluc

[0062] In order to obtain a lentiviral plasmid stably expressing IFN-β-Gluc, the vector pHAGE-bsd and the synthetic IFN-β-Gluc fragment (SEQ ID NO: 1) were double-treated with restriction endonucleases Spe I and Xba I, respectively. Enzyme digestion, the EF-1α promoter and downstream CDS insertion site in the digested linear vector were ligated with the IFN-β-Gluc fragment through homologous recombination, the ligated product was transformed into competent cells, and detected by bacterial liquid PCR Positive colonies, expanding culture and extracting plasmids, the construction process is as follows Figure 1A shown;

[0063] The extracted plasmid was digested and identified by restriction endonucleases Spe I and Xba I, and the electrophoresis results of the digested products were as follows: Figure 1B As shown, two bands of about 6800bp and 800bp were generated after double digestion with Spe I...

Embodiment 2

[0065] Example 2 lentiviral packaging

[0066] HEK-293T cells in the logarithmic growth phase were inoculated in a 10cm culture dish, and the cell density was about 2×10 5 cells / mL; after 24 hours, when the cell density reaches 80%, replace the medium with Opti-MEM;

[0067] Use Lipo2000 to transfect the constructed recombinant expression vector pHAGE-IFN-β-Gluc and helper plasmid, the amount of transfection is 12 μg for pMD2.G, 10 μg for psPAX2, and 22 μg for pHAGE-IFN-β-Gluc; culture for 8 hours, replace the culture The base is 10% FBSDMEM, continue to culture for 24 hours, and observe the state of the cells;

[0068] Collect the supernatant once at 48h and 72h respectively, mix the supernatant obtained twice, centrifuge at 1000rpm for 5min, filter through a 0.22μm microporous membrane to obtain lentivirus, and store it at -80°C for later use.

Embodiment 3

[0069] Example 3 Screening of cell lines stably expressing IFN-β-Gluc

[0070] Inoculate A549 cells in 6-well plates, grow to 60% on the second day, and carry out lentivirus infection;

[0071]Discard the supernatant before infection, wash the cells 1-2 times with sterile PBS, replace with lentiviral medium, and add 8 μg / mL polybrene to assist infection; 24 hours later, replace the medium with complete medium containing 10% FBS;

[0072] After 72 hours of infection, blasticidin (BSD) (10 μg / mL) was added to screen and culture polyclonal cells. After about 3 days, all cells in the uninfected control group died, and positive clones grew out of cells in the infected group; by limiting dilution Monoclonal cells were selected by this method, and the A549 cell line stably expressing IFN-β-Gluc was obtained after expansion.

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Abstract

The present invention provides a reporter gene cell line and a construction method and an application thereof. A genome of the reporter gene cell line is integrated with a chimeric gene and the chimeric gene comprises an IFN-beta promoter and a reporter gene; and the report gene is a secreted luciferase gene. The constructed reporter gene cell strain can secrete luciferase outside cells, providesa simple means for detecting activation of an IFN pathway, and lays a foundation for a further study of virus-induced IFN-beta transcription and regulation, and dynamic monitoring of IFN-beta promoteractivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a reporter gene cell line and its construction method and application, in particular to an IFN-β promoter-secreting luciferase stable expression cell line and its construction method and the detection of IFN pathway activation state application. Background technique [0002] Innate immunity is the first line of defense for cells against virus infection. After virus infects cells, the pattern recognition receptors (RIG-I, TLR3, cGAS, etc.) etc.), signaling proteins (TBK1, IKK, etc.), transcription factors (NF-κB, IRF-3, etc.) transcribe pro-inflammatory cytokines and interferon (IFN), in which IFN interacts with cell surface through autocrine or paracrine pathways IFN receptor (IFNR) binds, activates the downstream JAK-STAT pathway, promotes the expression of a large number of interferon-stimulated genes, and exerts antiviral effects. IFN is a secreted glycoprotein, and its family mem...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N9/02C12N15/867C12N5/10G01N33/68G01N33/569
CPCC07K14/565C12N5/0613C12N9/0071C12N15/86C12N2510/00C12N2740/15043C12Y114/14003G01N33/56966G01N33/68
Inventor 孙英杰丁铲仇旭升廖瑛谭磊孟春春宋翠萍王飒于圣青
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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