摘要
Toll样受体4(Toll-like receptor 4,TLR4)在机体的免疫反应中发挥重要作用,该基因突变会影响受体的信号转导能力和机体的疾病抗性/易感性。文章在前期工作的基础上,进一步分析c.611 T>A(p.Leu204His)、c.1027C>A(p.Gln343Lys)和c.1605 G>T(p.Leu535Phe)3个错义突变对猪TLR4功能的影响。利用RT-PCR方法克隆猪TLR4基因全长编码区并引入定点突变;利用真核表达、双荧光素酶报告系统和Western blotting方法在瞬时转染的PK-15细胞内研究3个单核苷酸多态(Single nucleotide polymorphisms,SNPs)对猪TLR4配体识别和信号转导能力的影响;同时,利用创造酶切位点PCR-RFLP方法分析对TLR4活性有显著影响的点突变在民猪、大白、长白和中国东北野猪4个群体中的分布。结果,成功获得了民猪TLR4基因的全长编码区和3个单碱基变异体,构建了不同等位基因的真核表达载体,在PK-15细胞内确定了c.1605 G>T变异导致TLR4向下游传递信号的能力显著降低(P<0.01),该SNP只存在于民猪和野猪中且频率较高。猪TLR4基因c.1605 G>T变异影响Toll样受体的信号传递,可能和机体的疾病抗性/易感性有关。
Toll-like receptor 4（TLR4） plays an important role in immune response and the polymorphism in it might affect protein signaling and host resistance/susceptibility to disease.This study was designed to characterize the functional relevance of 3 nonsynonymous single nucleotide polymorphisms（SNPs）,c.611 T〉A（p.Leu204His）,c.1027 C〉A（p.Gln343Lys）,and c.1605 G〉T（p.Leu535Phe）,which were selected based on our previous studies.RT-PCR method was used to clone the complete coding sequence of porcine TLR4 gene and the PCR-based method was used to introduce the point mutation.The effects of 3 SNPs on the ligand recognition and signaling of porcine TLR4 were investigated in transiently transfected PK-15 cells using dual-luciferase reporter system and Western blotting method.At the same time,the distribution of c.1605 G〉T among pig populations composed of Min pig,Yorkshire,Landrace,and Wild boar from northeastern China was studied by created restriction site PCR-RFLP method.The complete coding sequence of TLR4 gene in Min pig and 3 variants with single point mutations were obtained.Eukaryotic expression vectors containing different alleles of porcine TLR4 were constructed.SNP c.1605 G〉T significantly decreased the TLR4 signaling（P〈0.01） and the polymorphism only existed in Min pig and Wild boar from northeastern China with high frequencies.SNP c.1605 G〉T in porcine TLR4 might affect the receptor function and host resistance/susceptibility to diseases
出处
《遗传》
CAS
CSCD
北大核心
2012年第8期1050-1056,共7页
Hereditas（Beijing)
基金
黑龙江省教育厅科学技术研究项目(编号:11551033)资助
