Dual-luciferase reporter gene vector of human TLR4 gene 3' untranslated region and building method and application thereof

Abstract

The invention discloses a dual-luciferase reporter gene vector of human TLR4 gene 3' untranslated region and a building method and application thereof and belongs to the field of molecular genetics. The dual-luciferase reporter gene vector is pGLTlr4 / PSV40 / 3UTR, and a DNA sequence of the vector is shown as SEQ ID NO.5; the vector comprises a human TLR4 gene 3' untranslated region sequence which isshown as SEQ ID NO: 3 and cloned to luc gene downstream in a dual-luciferase reporter vector pGL3-Promoter. The vector can be used for detecting impact of SNP sites on the TLR4 gene 3' untranslated region on TLR4 gene expression and detecting targeting relation between the SNP sites on the TLR4 gene 3' untranslated region and miRNA.

Description

technical field [0001] The invention discloses a dual-luciferase reporter gene carrier of the 3' untranslated region of 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 p...

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 full-length 3'-UTR of most target genes (ranging from hundreds to thousands of bases), there are many identical restriction enzyme sites The points interfere with each other, so the traditional luciferase reporter gene vector can only insert a small DNA sequence (200 to 300 bases) in the 3'-UTR into the commercial luciferase reporter vector by enzyme-cut ligation. In the multiple cloning site, the construction of the full-length sequence cannot be realized; secondly, after the insert fragment is inserted into the multiple cloning site of the commercial luciferase reporter vector by enzyme digestion and ligation, it is usually inserted between the luciferase luc gene and the target A redundant sequence such as a restriction endonuclease recognition site remains between the inserted fragments, which may affect the accuracy of the experimental results; finally, because the traditional luciferase reporter vector is not inserted into the untranslated region of the complete target gene, it is only A short sequence was intercepted and constructed downstream of the luciferase gene. This structural composition is quite different from the real situation in vivo, so the selected regulatory factors often cannot play a role in the physiological state in vivo, just as Su Xiaoping et al. through miRIP The method found that only a small number of interacting miRNAs screened by the traditional luciferase reporter gene system were identical to the real situation in endosomes

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