猪Toll样受体4基因(TLR4)外显子SNP检测及生物信息学分析

摘要

Toll样受体4(Toll-like receptor4,TLR4)是天然免疫系统识别病原微生物的主要受体,其多态性与动物对相关病原的免疫力有着明显的相关性.为了对猪TLR4基因进行多态性分析,试验采用PCR-SSCP结合核苷酸测序法研究了梅山猪、二花脸猪、苏钟猪、姜曲海猪、淮猪、金华猪、大约克猪、皮特兰猪及金华与皮特兰杂交F2代猪( JPF2)9个群体共90个样本TLR4基因外显子序列的单核苷酸多态性,并对猪TLR4基因进行了生物信息学分析.结果检测到4个突变:G400A、G962A、C1027A、G960A,其中前3个为有义突变,且C1027A突变引起编码氨基酸性质的改变；生物信息学分析表明猪TLR4基因含有一个2526 bp的开放阅读框,编码841个氨基酸；序列比较发现猪与牛、绵羊、人和小鼠TLR4基因cDNA序列相似性分别为87％、86％、79％、73％,编码的氨基酸序列相似性分别为81％、80％、73％、64％.TLR4基因多态性检测结果表明猪TLR4基因的单核苷酸多态性在中外猪种中存在明显差异,结合生物信息学分析推测该基因编码蛋白质LRR(富含亮氨酸重复序列)功能域上的突变可能与猪种对疾病的易感性不同有关.%As a significant pattern-recognition receptor, Toll-like receptor 4 (TLR4) protein mainly recognizes the pathogenic microorganism in innate immune system, and its polymorphism has a profound influence on response to a wide range of pathogens and is associated with resistance and susceptibility to diseases. In order to study the polymorphisms,porcine TLR4 gene exon was detected by PCR-SSCP of 90 samples in 9 pig populations, including Meishan, Erhualian, Shuzhong, Jiangquhai, Huai, Jinhua, Large Yorkshire, Pietrain and JinhuaxPietrain hybrid F3(JPF2). Meanwhile, the gene sequence characters were analyzed by bioinformatics. Hie results showed 4 SNP sites, including G400A, G960A C962A and C1027A were detected and the first three were no-synonymous SNP sites. C1027A changed the character of amino acid. The bioinformatics a-nalysis revealed that porcine TLR4 gene contained an open reading frame of 2 S26 bp, encoding 841 amino acids(aa). The nucleotide sequence of TLR4 cDNA showed 87% , 86% , 79% and 73% similarties with those of cattle, sheep, human and mouse, respectively, and amino acid sequence showed 81% , 80% , 73% and 64% similarties, respectively. They existed marked differences in single nucleotide polymorphisms (SNPs) of porcine TLR4 gene between western and oriental breeds. By bioinformatics analysis, it was predicted that the mutations in LRRs domain of porcine TLR4 gene might be associated with resistance and susceptibility to diseases.