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

A dual-luciferase, non-translated region technology, applied in chemical instruments and methods, biochemical equipment and methods, introduction of foreign genetic material using vectors, etc. problems, to achieve the effect of helping the research process and speeding up the research process

Inactive Publication Date: 2019-10-25
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 an...

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: A dual-luciferase reporter gene carrier of the 3' untranslated region of the human source TLR4 gene, the dual-luciferase reporter gene carrier is pGLTlr4 / PSV40 / 3UTR, and the DNA sequence of the carrier is shown in SEQ ID NO.5 ;

[0035] The dual luciferase reporter gene vector contains the sequence of the 3' untranslated region of the human TLR4 gene, the sequence of the 3' untranslated region of the human TLR4 gene is shown in SEQ ID NO: 3, and the sequence of the 3' untranslated region of the human TLR4 gene is cloned into Downstream of the luc gene in the dual luciferase reporter vector pGL3-Promoter;

[0036] The construction method of the dual-luciferase reporter gene carrier of the 3' untranslated region of the human source TLR4 gene, the specific steps are as follows:

[0037] (1) Referring to the DNA sequence of the human TLR4 gene in GeneBank (accession number NG_011475) and the sequence of the pGL3-Promoter luciferase reporter vector, use the pr...

Embodiment 2

[0084] Example 2: Application of dual luciferase reporter gene vector pGLTlr4 / PSV40 / 3UTR (for detecting the impact of SNP sites on the 3' untranslated region of TLR4 gene on TLR4 gene expression)

[0085] Determine the SNP site to be studied

[0086] By using the Ferret-master genetic variation information analysis tool, combined with the review of relevant data, rs7873784 (G>C) and rs7873784 (G>A) were determined as the SNP sites to be studied that may affect the expression of the TLR4 gene;

[0087] Construction of mutant vectors

[0088] According to the method described in the document "Method for Realizing Site-Directed Mutagenesis on Circular DNA Molecules Larger than 10kb" with application number 201711203895.9, based on the dual luciferase reporter gene vector pGLTlr4 / PSV40 / 3UTR in the corresponding rs7873784 (G>C) and Site-directed mutation was performed on the rs7873784(G>A) site, and two mutation vectors, mut / rs784_G>C and mut / rs784_G>A, were constructed; all mutat...

Embodiment 3

[0098] Example 3: Application of dual luciferase reporter gene carrier pGLTlr4 / PSV40 / 3UTR (detection of the targeting relationship between the SNP site and miRNA on the 3' untranslated region of the TLR4 gene)

[0099] Simultaneous screening of three miRNA-related databases using bioinformatics methods: http: / / www.bioguo.org / miRNASNP2 / , http: / / www.targetscan.org / vert_71 / and https: / / snpinfo.niehs.nih .gov / snpinfo / , it is predicted that miRNA hsa-let-7d-3p can target the 3'-UTR of TLR4 gene, the binding target site is "TCGTATA", and rs7873784 is located in the underlined base G; when rs7873784 (G>A) After the mutation, only 6nt of the miRNA can bind to the target site. This mutation may cause the miRNA to lose its ability to bind to the 3'UTR of the TLR4 gene (see Figure 9 );

[0100] Co-transfection and dual luciferase activity assay

[0101] Spread HEK293T cells at 4×104 per well on a 24-well plate one day before transfection, and add complete medium (DMEM medium plus 10% ...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/52C12Q1/66
CPCC07K14/705C12N15/52C12N15/66C12N15/85C12Q1/66
Inventor 郑冰蓉陈国栋叶汉风杨红菊
Owner YUNNAN UNIV
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