用PCR-SSCP方法检测猪Toll样受体4(TLR4)基因外显子3SNP.docVIP

用PCR-SSCP方法检测猪Toll样受体4（TLR4）基因外显子3的SNP 周　波,刘传武，虞德兵，黄瑞华，刘红林，王林云 （南京农业大学动物科技学院　江苏南京　210095） 摘要：Toll样受体(TLRs)能识别各种微生物成分并诱导免疫反应。作为TLR家族中一员的TLR4是识别革兰氏阴性菌内毒素—脂多糖（LPS)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。本文利用PCR-SSCP并结合PCR产物双向测序的方法，对梅山猪、新淮猪、大白猪、长白猪和杜洛克猪共203个样本TLR4基因外显子3部分片段的单核苷核多态性进行了研究。结果检测到了5个SNP，分别是T611A，G826A，G960A，G962A和C1027A，其中有4个非同义的SNP，有2个SNP的氨基酸性质发生了改变。猪TLR4的SNP出现频率在各猪种中有差异，其中T611A仅在新淮猪和大白猪中检测到，而G826A和C1027A仅分别在新淮猪和大白猪中检测到。 关键词：猪；TLR4；PCR-SSCP；SNP Detecting SNP in porcine TLR4 gene with polymerase chain reaction- single strand conformation polymorphism (PCR-SSCP) ZHOU Bo, LIU Chuan-wu, YU De-bing，LIU Hong-lin,WANG Lin-yun (College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China) Abstract: Toll-like receptors (TLRs) recognize various microbial components and induce immune responses. TLR4, a member of TLRs, functions as the transmembrane component of LPS receptor complex, and its polymorphisms and/or differences have a profound influence on responses to a wide range of pathogens and are associated with resistance and susceptibility to diseases. In this study, 4 sequence fragments of TLR4 gene exon 3 in 5 strains of pigs was detected by PCR-SSCP and sequencing. The strains involved were Meishan, Xinhai, Large White, Landrace and Duroc pigs, and the numbers of them were 30, 50, 106, 2 and 15, respectively. Five SNP sites were detected, including T611A，G826A，G960A，G962A and C1027A, 4 of which were nonsynonymous SNPs, and 2 of which changed the character of amino acid. The frequencies of SNPs detected were difference among breeds. SNP T611A was detected only in Xinhuai and Large White pigs; SNP G826A and C1027A were detected only in Xinhuai and Large White pigs, respectively. Keywords: pig; TLR4；PCR-SSCP；SNP 当前，猪的传染病已成为现代养猪生产需要解决的主要问题。例如，在我国猪喘气病(猪地方流行性肺炎，swine enzootic pneumonia, SEP)和萎缩性鼻炎(atrophic rhinitis, AR)的流行每年都给养猪业带来了巨大的经济损失[1]。一般，猪喘气病的病原猪肺炎支原体(Mycoplasma hyopneumoniae, M. hyo)和猪萎缩性鼻炎的病原体支气管败血波氏杆菌(Bordetella bronchiseptica)和产