Dual-luciferase reporter gene carrier based on human TLR4 gene promoter region and construction method and application of dual-luciferase reporter gene carrier

Abstract

The invention discloses a dual-luciferase reporter gene carrier based on a human TLR4 gene promoter region and a construction method and application of the dual-luciferase reporter gene carrier, and belongs to the field of molecular genetics. By designing a primer TPprimer 1 and a primer TPprimer 2 with the 5' tail ends including homologous arms in the upstream and downstream sequences of an interface, the -3494 sequence to the +235 sequence of a TLR4 gene are obtained through amplification, the -3494 sequence to the +235 sequence of the TLR4 gene are seamlessly cloned to NcoI loci of a dual-luciferase reporter carrier pGL3-basic through a Gibson assembling method so as to construct the dual-luciferase reporter gene carrier pGLTlr4 / -3494+235 in the promoter region at the 5' end of the humanized TLR4 gene. The dual-luciferase reporter gene carrier can be digested through restriction enzymes such as BglII, KpnI and EcoRI and then reconnected to construct the derivative reporter gene carrier including promoters with different lengths, thus the derivative reporter gene carrier is used for analyzing the promoter sequence of the TLR4 gene in cells of different tissue sources, and specificity regulating and controlling mechanisms for promoter cells of the TLR4 gene and the like are investigated.

Description

technical field [0001] The invention discloses a dual-luciferase reporter gene carrier based on the human TLR4 gene promoter region, 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...

AI Technical Summary

Problems solved by technology

However, this method is not suitable for the insertion of large fragments, because there are many identical restriction enzyme sites that interfere with each other, and there are often no available endonucleases

In addition, the method of enzyme-cut ligation will leave a piece of redundant sequence such as a restriction endonuclease recognition site between the luciferase luc gene and the target insert fragment, which may affect the accuracy of the experimental results

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