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

用PCR-SSCP方法检测猪Toll样受体4（TLR4）基因刘传武摘要：革兰氏阴性菌内毒素脂多糖（LPS)的主要受体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、长白猪杜洛克TLR4基因关键词：TLR4；PCR-SSCP；SNPDetecting 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 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)和产毒素多杀性巴氏杆菌asteurella multocida)会长期存留在猪场，很难彻底清除。因此，从猪的抗病育种上增强猪对以上顽固病原的抵抗力，会是控制这些传染病的一个较好的有效途径。 研究表明，免疫分子Toll样受体家族（Toll-like receptor, TLRs）TLR4是TLRs中的一员是天然免疫识别机制革兰氏阴性菌表达内毒素脂多糖（Lipopolysaccharide-LPS)应答的主要受体。许多证据显示TLR4在LPS跨膜信号转导中起主导作用TLR4基因碱基的突变会导致宿主对革兰阴性菌的反应能力严重丧失。single nucleotide polymorphism，SNP)。Hiroki Shinkai等研究也表明，11个猪种TLR4基因中的7个氨基酸的替换全部位于外显子3中[5]。聚合酶链反应-单链构象多态性分析（Single Strand Conformation Polymorp