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NF-kappa B dual-luciferase reporter cell capable of stably expressing pig-origin RIG-I receptor genes 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 lack of research content of livestock natural immune receptor RIG-I and the absence of porcine RIG-I signaling pathway Research reports and other issues

Inactive Publication Date: 2018-03-23
YANGZHOU UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The research content of domestic animal natural immune receptor RIG-I is lacking, and there is no research report on porcine RIG-I signaling pathway. It is necessary to establish a cell system for porcine RIG-I signaling pathway

Method used

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  • NF-kappa B dual-luciferase reporter cell capable of stably expressing pig-origin RIG-I receptor genes and construction method thereof
  • NF-kappa B dual-luciferase reporter cell capable of stably expressing pig-origin RIG-I receptor genes and construction method thereof
  • NF-kappa B dual-luciferase reporter cell capable of stably expressing pig-origin RIG-I receptor genes and construction method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0037] (1) Construction of pcDNA eukaryotic expression vector containing pRIG-I coding gene

[0038] Amplification and cloning of the coding region of the pRIG-I gene.

[0039] A pair of cloning PCR primers were designed, the upstream primer was ttcgcGTCGACatgacagcagagcagcggcggaatc (SEQ ID NO.1); the downstream primer was ttcgcCCCGGGctcaaggttgcccattccctgaagacccag (SEQ ID NO.2). Total RNA was extracted from porcine PK15 cells, and cDNA was generated by reverse transcription (RT). Using cDNA as a template, the coding region of pRIG-I gene was amplified by PCR, and a 2.8kb DNA fragment was amplified. The amplified PCR fragment was double digested with SalI and SmaI, and the Gateway pENTR4 intermediate entry vector with the C-terminal 2XHA expression tag was ligated with T4 ligase. The ligated product was transformed into competent TOP10, and the transformed bacterial TOP10 was coated with kanamycin-resistant (Kanamycin) bacterial agar plate, the recombinant clone was identified ...

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Abstract

The invention belongs to the technical field of biology and specifically relates to an NF-kappa B dual-luciferase reporter cell system capable of stably expressing pig-origin RIG-I receptor genes anda construction method thereof. The cell system is an HEK293-NF-kappa B cell which the pig-origin RIG-I (pRIG-I) receptor genes are transferred into, and the HEK293-NF-kappa B cell is an HEK293 cell which NR-kappa B firefly luciferase and internal reference renilla luciferase are guided into. The stable cell system disclosed by the invention can be applied to researching detection on downstream NF-kappa B promoter reporter genes activated by pRIG-I. The invention further provides a biological material for an RNA-Seq technology to screen stable cell internal differential expression genes under silent and active states of the pRIG-I in high throughput; furthermore, a foundation and a platform are established for further researching species specificity of the pRIG-I.

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 pig-derived RIG-I (pRIG-I) receptor gene and a construction method thereof. More specifically, the present invention provides a stable cell line that can be used to detect pRIG-I activation and activate a downstream NF-κB promoter reporter gene detection. The present invention also provides biological materials for high-throughput screening of pRIG-I silent and activated differentially expressed genes in stable cells by RNA-Seq technology, and lays a foundation and platform for further research on the species specificity of RIG-I. Background technique [0002] RIG-like receptors (RLRs) RIG-I [0003] RLRs are the major viral RNA receptors in the cytoplasm of cells. RIG-I was first discovered by screening cDNA libraries [1,2] , other members also include MDA5 and LGP2. RIG-I and MDA5 have similar structural ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/12
CPCC12N5/0686C07K14/70596C12N5/0603C12N15/85C12N2510/00C12N2800/107
Inventor 朱建中李双洁何姗周荣云陈南华朱美芹
Owner YANGZHOU UNIV
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