NF-kB dual-luciferase reporting cell line for stably expressing human TLR8 receptor gene and construction method thereof

A dual-luciferase, stable expression technology, applied in the field of NF-κB dual-luciferase reporter cell line and its construction, can solve the problem of reducing the sensitivity of tumor cells to chemotherapy drugs

Inactive Publication Date: 2018-03-23
YANGZHOU UNIV
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Problems solved by technology

Although the effects of TLR8 stimulators on tumor cells vary due to different tumor cell types, the activation of TLR8 can activate the NF-κB transcription factor, thereby upregulating the expression of the apoptosis protein Bcl-2, and promoting the proliferation and survival of tumor cells At the same time, it reduces the sensitivity of tumor cells to chemotherapy drugs and produces chemotherapy resistance [8,9]

Method used

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  • NF-kB dual-luciferase reporting cell line for stably expressing human TLR8 receptor gene and construction method thereof
  • NF-kB dual-luciferase reporting cell line for stably expressing human TLR8 receptor gene and construction method thereof

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Experimental program
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Effect test

Embodiment Construction

[0029] (1) Construction of pcDNA3.1(-) eukaryotic expression vector containing hTLR8 gene:

[0030] Using the cDNA obtained by reverse transcription of total RNA extracted from human mesenteric lymph nodes as a template, using specific primers (upstream primer: ttcgcGCTAGCatggaaaacatgttccttcagtcg (SEQ ID NO.1); downstream primer: gttcgcGATATCgtattgcttaatggaatcgacatacatatattg (SEQ ID NO.2); upstream and downstream primers Respectively with Nhe I and EcoR V restriction sites) to amplify the full-length coding region of hTLR8, after purification, the PCR product is digested with restriction endonucleases Nhe I and EcoR V, and the same restriction endonuclease with the C-terminal HA tag After ligation of pcDNA3.1(-), the competent DH5α cells were transformed. 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 hTLR8 gene was obtained. Aft...

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Abstract

The invention belongs to the technical field of biology and particularly relates to an NF-kB dual-luciferase reporting cell line for stably expressing human TLR8 receptor gene and a construction method thereof. The cell line is HEK293-NF-kB cells with transgenosis of the human TLR8 gene, and the HEK293-NF-kB cells are HEK293 cells with introduction of NF-kB luciferase and internal reference ranilla luciferase. The NF-kB dual-luciferase reporting cell line and the construction method also provide a biological material for studying the action of TLR8 ligands in tumor treatment and utilizing an RNA-Seq technology to screen genes for stabilizing differential expression in the cells under the silent and activated states of hTLR8 by high flux, and lays foundation and platform for further studying the species specificity of the TLR8.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, it relates to a NF-κB dual-luciferase reporter cell line stably expressing human TLR8 (hTLR8) receptor gene and a construction method thereof. More specifically, the present invention provides a stable cell line that can be used to study hTLR8 activation and activate downstream NF-κB promoter reporter gene detection. The present invention also provides biological materials for the study of the role of TLR8 ligands in tumor therapy, as well as the high-throughput screening of hTLR8 silent and active states in stable intracellular differentially expressed genes by using RNA-Seq technology, and provides a basis for further research on the species of TLR8 Specificity lays the foundation and platform. Background technique [0002] As an important component of pattern recognition receptors (Pattern recognition receptors, PRR), Toll-like receptors (Toll-like receptors, TLR) play a vital role...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/12C12R1/91
CPCC07K14/70596C12N5/0603C12N5/0686C12N15/85C12N2510/00C12N2800/107
Inventor 朱建中夏芃芃李双洁敖大陈南华
Owner YANGZHOU UNIV
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