Effect Of TLR4 Promoter Polymorphisms On Transcription Activity In Neonates

[Objective] To reveal the effect of TLR4 promoter polymorphisms on transcription activity in neonates and analyze the relationship between SNPs in TLR4 promoter and neonatal G-bacteria infection.[Methods] Selected the SNPs in TLR4 gene promoter in neonatal population, which maybe influence TLR4 gene expression through our earlier studies.Including A-2604G,A-2570G,T-2431C,G-208,A-2026G,T-1607C. Choose No.50 collection DAN of TLR4 as the template for PCR(polymerase chain reaction),the aimed six SNPs in TLR4 gene promoter of which are homozygotes as-2604AA,-2570AA,-2431TT,-2081GG,-2026AA,-1607TT.To study the function of the SNPs, we first constructed basic Pg13-TLR-4 promoter plasmid. And obtained different promoter plasmids by rite-directed mutagenesis technique, then measured TLR4 promoter activity by Dual-Luciferase Reporter assay system.[Results] 1,Construction and assessment of the basic pGL3-TLR4 promoter plasmid:Using PCR technique to amplification of the aimed TLR4 DNA fragment, and recombinanted the PCR production to the plasmid PMDT-18. Assessment the production by bacterial colony PCR. There is a production at equal of expected fragment at about 2600 bp. Then constructed the enzyme cutting production to pGL3 plasmid. Electrophoresis assessment shows the fragment is to fit our expected. Sequencing test reports that the fragment's base in pGL3-TLR4 promoter plamid we constructed is completely correct as in data base.2,Construction and assessment of the mutagenic pGL3-TLR4 promoter plasmid:-2604GG,-2570GG,-2431CC,-2081 AA,-2026GG,-1607CC:Design the primers of mutational sites, and take the constructed pGL3-TLR4 promoter plasmid as the template. Using the kit of site-directed mutagenesis to carry out PCR.And we got PCR production of -2604GG,-2570GG,-2431CC,-2081AA,-2026GG,-1607CC mutagenic pGL3-TLR4 promoter plasmids, Electrophoresis after dual enzymes cutting indicate the strap are expected. Sequencing test certify that we get the mutant plasmid genotype as-2604GG,-2570GG,-2431CC,-2081AA,-2026GG,-1607CC. 3,Effect of TLR4 promoter mutants(SNP) on transcription activity:Dual-Luciferase Reporter assay system shows that-2431 CC promoter and-2026GG promoter have a lower activity than-2431TT promoter and-2026AA promoter, it implys that-2431T→C and-2026 A→G base variation can reduce the activity of TLR4 promoter. There are no significant difference of promoter activity between-2604AA promoter and-2604GG promoter,-2570AA promoter and-2570GG prmoter,-2081GG promoter and-2081 AA promoter,-1607TT promoter and-1607CC promoter.[Conclusion 1-2431 T→C and-2026A→G base variation can reduce the transcription activity of TLR4 promoter. While-2604A→G,-2570A→G,-2081G→A,-1607T→C base variation have no impact on transcription activity of TLR4 gene promoter.