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[Objective] reveals neonatal TLR-4 gene promoter region SNP loci biological significance is further analyzed from the perspective of gene polymorphism SNP loci base variation of target gene expression, explore the the TLR4 promoter district SNP provide the basis and neonatal G-bacteria infection. [Method] In this study, specific amplification by PCR containing sites required TLR-4 gene promoter region of the DNA fragment, build PGL3-TLR-4 promoter foundation plasmid containing the target SNP site-directed mutagenesis promoter each plasmid plasmid transfected HEK293 cells and test the efficiency of transfection, transcriptional activity observed TLR4 gene promoter region SNP dual luciferase reporter gene system. [Results] 1.pGL3-TLR4 promoter plasmid and identification: six SNP loci TLR4 gene promoter fragment count the 2697bp (-2 694 - 3) from the purpose of the genome by PCR amplification, the PCR foundation The product was ligated recombinant PMDT-18 vector, colony PCR product was subjected to agarose gel electrophoresis, and the product is located between 2000bp-5000bp, approximately 3000bp, consistent with experimental expected fragment size. Will the product digested build to a fluorescent reporter system vector pGL3. Electrophoresis preliminary identification of plasmid was constructed successfully. Sequencing, pGL3-TLR4 construct plasmid insert sequence, base sequence is entirely consistent with the database. 2.-2604GG,-2570GG,-2431CC,-2081AA,-2026GG,-1607CC mutant plasmid Construction and identification of: basic plasmid construct pGL3-TLR4 promoter as a template, were used-2604A-2570A-2431T - 2081G,-2026A,-1607T locus mutation primers and site-directed mutagenesis kit reaction site-directed mutagenesis, site-directed mutagenesis for mutant plasmids. The sequencing results proved to be successful-2604GG,-2570GG,-2431CC,-2081AA,-2026GG,-1607CC mutant plasmids. Dual luciferase reporter gene system testing: selection of HEK293 cells as transfected cells, plasmid transfection of eukaryotic expression vector (pCY/U6/GFP/Neo) simultaneously transfected with GFP fluorescence marker to observe the transfection of HEK293 efficiency. Transfection 48H, to a large number of green fluorescent protein expression was observed. Dual luciferase reporter system: Compared with pGL3-TLR4 basic promoter plasmid -2431 sites by T mutation is C -2026 loci A mutation G, promoter activity decreased, and -1607 loci T to C -2 081 sites by G mutated to A, -2570 locus mutation from A to G, -2604 locus mutation G by A, of TLR4 promoter activity did not change significantly. [Conclusion] -2431 locus T mutation is C -2026 locus mutation from A to G, the promoter activity decreased, we speculate that the the -2431 locus by T mutation of C -2026 loci by A mutation G nucleotide variation caused lower promoter activity may eventually lead down target gene expression, thus affecting TLR4 reaction to LPS, to change the body susceptibility to infection and the severity of post-infection.
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