猪TLR3基因A1116T点突变功能初步分析

A1116T SNP Functional Analysis of Porcine TLR3 Gene

为了确定猪TLR3基因A1116T错义突变的生物学效应,研究其与猪疾病抗性/易感性的关系,本研究采用重叠延伸PCR和定点突变技术构建不同等位基因的真核表达载体,采用双荧光素酶检测分析系统及Real-timePCR方法在体外培养的细胞中研究不同等位基因在信号转导中的作用。成功地构建了野生型和突变型TLR3的真核表达载体,双荧光素酶检测分析表明突变型活化转录因子NF-κB、激活ISRE的能力都减弱;Real-time PCR分析表明突变型诱导转录IL-6的能力下降。结果表明该点突变影响猪TLR3的信号转导能力。

In order to reveal the biological role of nonsynonymous SNP A1116T of porcine TLR3 gene and to investigate the relationship of the SNP with disease resistance/susceptibility,the study was designed to construct eukaryotic expression vector of two types of alleles by splicing overlap extension PCR and site-directed mutation methods,and to analyze the function of the two alleles in signal transduction in cultured PK-15 by dual-luciferase reporter assay and Real-time PCR methods.Two types of eukaryotic expression vectors of porcine TLR3 were successfully constructed.The dual-luciferase reporter assay demonstrated that the mutant type TLR3 allele was defective for activating TLR3-dependent reporter constructers.The real-time PCR analysis showed that the mutant type was reduced in inducing IL-6 transcription.The results indicate that the mutation influences the signal transduction ability of porcine TLR3.