Multifunctional dual luciferase reporter gene vector based on human TLR4 gene, construction method and application thereof

Abstract

The invention discloses a multifunctional dual luciferase reporter gene vector based on human TLR4 gene, a construction method and an application thereof, which belong to the field of molecular genetics. The multifunctional dual luciferase reporter gene vector of the present invention is pGLTlr4 / -3494+235 / 3UTR, and a DNA sequence of the vector is shown as SEQ ID NO.5; the multifunctional dual luciferase reporter gene vector comprises the human TLR4 gene-3494 to +235 sequence and the complete 3'-UTR sequence, the human TLR4 gene-3494 to +235 sequence is shown as SEQ ID NO: 1, and the complete 3'-UTR sequence is shown as SEQ ID NO: 2; and the human TLR4 gene-3494 to +235 sequence and the 3'-UTR complete sequence are cloned into upstream and downstream of the firefly luciferase luc gene in the pGL3-Basic plasmid respectively. The method seamlessly clones the upstream and downstream non-coding sequences of the TLR4 gene into the upstream and downstream of the firefly luciferase luc gene, respectively, the structural composition of the vector is closer to the real situation than the traditional luciferase reporter plasmid, and the negative effect of an exogenous promoter on the experimental results is effectively avoided.

Description

technical field [0001] The invention discloses a multifunctional dual-luciferase reporter gene carrier based on human TLR4 gene, its construction method and application, and belongs to the field of molecular genetics. Background technique [0002] Toll-like receptors (Toll-Like receptors, TLRs) are pattern recognition receptors (pattern recognition receptors, PRRs), which are portal proteins for inflammatory signal transmission, and will trigger cell signal transduction after binding to their corresponding ligands under physiological conditions. It causes the high expression of many downstream genes related to inflammatory response, promotes the release of various inflammatory cytokines, thus plays a vital role in the process of fighting against invading pathogens, and can effectively activate innate and acquired immune responses. Among them, TLR4 is the first discovered Toll-like receptor protein, which belongs to type I transmembrane protein. It consists of three parts: ex...

AI Technical Summary

Problems solved by technology

Reporter gene vectors constructed by traditional methods have the following defects: First, due to the long length of the promoter region and full-length 3'-UTR of most target genes (ranging from hundreds to thousands of bases), there are many same limitations Therefore, the traditional luciferase reporter gene vector can only insert a small piece of DNA sequence (200 to 300 bases) in the promoter region or 3'-UTR into the In the multiple cloning site of the commercialized luciferase reporter vector, the construction of the full-length sequence cannot be realized; secondly, after the insert fragment is inserted into the multiple cloning site of the commercialized luciferase reporter vector by restriction enzyme ligation, usually A redundant sequence such as a restriction endonuclease recognition site remains between the luciferase luc gene and the target insert, which may affect the accuracy of the experimental results; finally, because the traditional luciferase reporter vector is not inserted completely For the untranslated region or promoter region of the target gene, only a small sequence is intercepted and constructed upstream or downstream of the luciferase gene. This structural composition is quite different from the real situation in vivo, so the selected regulatory factors are often not available in vivo. It functions under physiological conditions, just as Su Xiaoping and others found through the miRIP method that only a very small number of interacting miRNAs screened by the traditional luciferase reporter gene system are the same as the real situation in endosomes

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