鸭嘴海豆芽MyD88基因克隆和鉴定

Identification and Characterization of MyD88 in Lingula Anatina

髓样分化因子88(myeloid differentiation factor 88,MyD88)是Toll样受体(Toll-like receptor)信号通路重要的上游接头分子。为丰富MyD88基因的系统进化数据,以原口动物鸭嘴海豆芽(Lingula anatina)为研究对象,克隆laMyD 88基因全长序列进行序列鉴定和进化分析,构建相关真核表达载体,通过细胞内荧光定位分析和报告基因实验验证laMyD 88在细胞中的定位及其功能。除此以外,分离鸭嘴海豆芽的不同组织,以及对鸭嘴海豆芽进行了细菌挑战试验,提取总RNA,通过实时荧光定量PCR对laMyD 88基因的组织表达谱和感染后急性期表达谱进行分析。成功克隆了鸭嘴海豆芽全长1695 bp的laMyD 88 cDNA序列,其开放阅读框(open reading frame,ORF)1320 bp,编码一个含439 aa的laMyD 88蛋白。所构建的真核表达载体pDesRed-laMyD 88在HeLa细胞中成功表达,细胞内定位特征同文昌鱼文献报道的一致,并且在HEK293T细胞中能剂量依赖性地激活NF-κB信号通路,表达谱分析表明laMyD 88基因在免疫相关组织表达量较高,并在细菌感染急性期上调表达。结果表明:laMyD 88在鸭嘴海豆芽的天然免疫应答过程中发挥着重要作用。研究可以为天然免疫信号通路的起源与进化研究提供比较数据。

Myeloid differentiation factor 88(MyD88)is an important upstream connector molecule of Toll-like receptor signaling pathway.In order to enrich the phylogenetic data of MyD88 gene in protostomes,this study cloned the full length sequence of laMyD 88 gene for sequence identification and evolutionary analysis,and constructed the related eukaryotic expression vector.The localization and function of laMyD 88 in cells were verified by intracellular fluorescence localization analysis and reporter gene assay.In addition,we isolated different tissues of lingula anatina,and carried out bacterial challenge test on lingula anatina,extracted total RNA,and analyzed the tissue expression profile of laMyD 88 gene and the expression profile of the acute stage after infection by real-time fluorescence quantitative PCR.We successfully cloned the full length 1695bp laMyD 88 cDNA sequence from lingula anatina,and its open reading frame(ORF)was 1320bp encoding a laMyD 88 protein containing 439 amino acids.The constructed eukaryotic expression vector pDesRed-laMyD 88 was successfully expressed in HeLa cells,and its intracellular localization characteristics were consistent with those reported in amphioxus.In addition,HEK293T cells could activate NF-κB signaling pathway in a dose-dependent manner.Expression profile analysis showed that laMyD 88 gene was highly expressed in immune-related tissues.The expression was up-regulated in the acute stage of bacterial infection.The results showed that laMyD 88 played an important role in the natural immune response of lingula anatina.This study can provide comparative data for studying the origin and evolution of natural immune signaling pathways.