NF-kappaB dual-luciferase report cell capable of stably expressing pig derived TLR8 receptor gene, and construction method of NF-kappaB dual-luciferase report cell

A dual-luciferase, stable expression technology, applied in the field of NF-κB dual-luciferase reporter cell line and its construction, can solve the problems of lack of research content on livestock TLR8 and no other reports on TLR8 signaling pathway

Inactive Publication Date: 2018-03-13
YANGZHOU UNIV
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Problems solved by technology

[0004] The research content of livestock TLR8 is lacking, and the TLR8 signaling pathway has no other reports except the applicant's previous published research. It is necessary to establish a cell system to study the porcine TLR8 signaling pathway

Method used

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  • NF-kappaB dual-luciferase report cell capable of stably expressing pig derived TLR8 receptor gene, and construction method of NF-kappaB dual-luciferase report cell
  • NF-kappaB dual-luciferase report cell capable of stably expressing pig derived TLR8 receptor gene, and construction method of NF-kappaB dual-luciferase report cell

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Embodiment Construction

[0027] (1) Construction of pcDNA3.1(-) eukaryotic expression vector containing pTLR8 gene (see literature MolImmunol.2008Jun; 45(11):3238-43):

[0028] Using the cDNA obtained by reverse transcription of total RNA extracted from porcine mesenteric lymph nodes as a template, the full-length coding region gene of pTLR8 was amplified by PCR using specific primers (the upstream and downstream primers had Nhe I and EcoR V restriction sites, respectively). After purification, the product was cleaved with restriction endonucleases Nhe I and EcoR V, ligated with pcDNA3.1(-) which was also cleaved with the C-terminal HA tag, and then transformed into competent DH5α cells. Transformed bacteria were cloned to extract the plasmid, and after the plasmid was identified correctly by enzyme digestion and sequencing, the pcDNA3.1(-) eukaryotic expression vector containing the pTLR8 gene was obtained. pcDNA3.1(-)-pTLR8 has a C-terminal HA expression tag. After transfection into HEK293 or COS-7 ...

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Abstract

The invention belongs to the field of biological technology, and in particular relates to an NF-kappaB dual-luciferase report cell line capable of stably expressing a pig derived TLR8 receptor gene, and a construction method of the NF-kappaB dual-luciferase report cell line. The cell line is an HEK293-NF-kappaB cell, in which a pTLR8 gene is transferred; and the HEK293-NF-kappaB cell is an HEK293cell, in which NF-kappaB luciferase and endodontic luciferase are introduced. The invention provides a stable cell line, which can be used for researching pTLR8 activated downstream NF-kappaB promoterreport gene detection. The invention also provides a biological material for an RNA-Seq technology to perform high-throughput screening on differential expression genes in the stable cell under the bTLR8 silent and activated states, and lays the foundation and a platform for further researching the species specificity of TLR8.

Description

technical field [0001] The invention belongs to the field of biotechnology. In particular, it relates to a NF-κB dual-luciferase reporter cell line stably expressing pig-derived TLR8 (pTLR8) receptor gene and a construction method thereof. More specifically, the present invention provides a stable cell line that can be used to study pTLR8 activation and activate downstream NF-κB promoter reporter gene detection. The present invention also provides biological materials for high-throughput screening of differentially expressed genes in stable cells of pTLR8 in silent and activated states by RNA-Seq technology, and lays a foundation and platform for further research on the species specificity of TLR8. Background technique [0002] Innate immunity is the first line of defense of the body's immune system against pathogenic infection, and is mediated by a series of pattern recognition receptors (PRR) that can recognize pathogen-associated molecule patterns (PAMP) in cells. Among...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/12
CPCC07K14/70596C12N5/0603C12N5/0686C12N15/85C12N2510/00C12N2800/107
Inventor 朱建中夏芃芃李双洁陈南华敖大
Owner YANGZHOU UNIV
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