NF-kB dual-luciferase reporter cell for stably expressing swine TLR5 receptor genes and construction method

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 porcine TLR5 function research reports, lack of research content of livestock TLR signal, lack of porcine TLR5 isolation and Identification of research reports and other issues

Inactive Publication Date: 2018-03-06
YANGZHOU UNIV
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Problems solved by technology

[0005] Although reported studies have investigated the function of porcine TLR5 using bacterial flagellin-stimulating and TLR5-blocking antibodies [4] , there are also reports on the functional identification of porcine TLR5 promoter [5] , but there is no report on the isolation and identification of porcine TLR5 itself
[0006] Livestock TLR signaling research content is scarce, and there is no functional research report on porcine TLR5 (pTLR5), so it is necessary to establish a cell system for porcine TLR5 signaling pathway

Method used

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  • NF-kB dual-luciferase reporter cell for stably expressing swine TLR5 receptor genes and construction method
  • NF-kB dual-luciferase reporter cell for stably expressing swine TLR5 receptor genes and construction method
  • NF-kB dual-luciferase reporter cell for stably expressing swine TLR5 receptor genes and construction method

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Embodiment Construction

[0030] (1) Construction of pcDNA eukaryotic expression vector containing pTLR5 encoding gene

[0031] Amplification and cloning of the coding region of the pTLR5 gene.

[0032] A pair of cloning PCR primers were designed, the upstream primer was ttcgcGTCGACatgggagactgcctggtcctgctcttg (SEQ ID NO.1); the downstream primer was ttcgcGATATCggagatggtcacgctttgcaactgaatgtcg (SEQ ID NO.2). Total RNA was extracted from porcine alveolar macrophages (PAM), and cDNA was generated by reverse transcription (RT). Using cDNA as a template, the coding region of pTLR5 gene was amplified by PCR, and a 2.6kb DNA fragment was amplified. The amplified PCR fragment was digested by SalI and EcoRV, and the Gateway pENTR4 intermediate entry vector with the C-terminal 2XHA expression tag was ligated by 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 ident...

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Abstract

The invention belongs to the technical field of biology and in particular relates to an NF-kB dual-luciferase reporter cell for stably expressing swine TLR5 receptor genes and a construction method thereof. A cell line refers to HEK293-NF-kB cells in which the swine TLR5 receptor genes are transferred, and the HEK293-NF-kB cells refer to HEK293 cells in which NF-kB firefly luciferase and renilla luciferase are transferred. The invention provides the stable cell line used in detection for researching pTLR5 activated downstream NF-kB promoter report genes. The invention also provides a biological material for performing high-throughput screening on differentially expressed genes in stable cells in quiesced and activated states in an RNA-Seq technology, and lays a foundation and platform forfurther researching species specificity of TLR5.

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 TLR5 (pTLR5) receptor gene and a construction method thereof. More specifically, the present invention provides a stable cell line that can be used to detect pTLR5 activation and activate a 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 pTLR5 in silent and activated states by RNA-Seq technology, and lays a foundation and platform for further research on the species specificity of TLR5. Background technique [0002] TOLL-like receptor type 5 (TLR5) [0003] As a type I transmembrane glycoprotein, TLR5 has an extracellular LRR domain, a transmembrane region and an intracellular Toll / IL-1R (TIR) ​​domain. TLR5 is the only TLR protein that recognizes bacterial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/65C12R1/91
CPCC07K14/70596C12N15/65C12N15/85
Inventor 朱建中周荣云李双洁何姗朱美芹朱媛媛
Owner YANGZHOU UNIV
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