斑马鱼myd88和traf6真核表达载体的构建  

Construction of Eukaryotic Expression Vectors for myd88 and traf6 from Zebrafish(Danio rerio)

作　　者：徐行 王子睿[1,2] 刘何奕 张颖 任建峰 李伟明[3] 张庆华[1,2] Xu Xing;Wang Zirui;Liu Heyi;Zhang Ying;Ren Jianfeng;Li Weiming;Zhang Qinghua(Key Lab of Ministry of Education for Exploration and Utilization of Aquatic Genetic Resources,Shanghai Ocean University,Shanghai,201306;National Pathogen Collection Center for Aquatic Animals,Shanghai,201306;Department of Fisheries and Wildlife,Michigan State University,East Lansing,USA,48824)

机构地区：[1]上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海201306 [2]上海海洋大学,国家水生动物病原库,上海201306 [3]密歇根州立大学渔业与野生动物系,东兰辛美国48824

出　　处：《基因组学与应用生物学》2021年第4期1488-1496,共9页Genomics and Applied Biology

基　　金：教育部留学回国人员科研启动基金项目(D-8002-15-0042);上海市教委重点创新项目(13ZZ127);中美海洋研究中心基金项目(A1-0209-15-0806)共同资助。

摘　　要：髓样分化因子88 (myeloid differentiation primary response gene 88, MYD88)和TNF受体相关因子6(TNF receptor associated factor 6, TRAF6)均属于Toll样受体(Toll-like receptors, TLR)家族成员中的关键衔接分子。斑马鱼是研究先天免疫应答的独特模式生物,为了构建myD88和traf6的真核表达载体,用于免疫应答机制的研究,克隆了斑马鱼myd88和traf6基因的编码区CDS全长序列,分别是855 bp和1 629 bp。结构分析表明,斑马鱼MYD88存在2个保守结构域为死亡结构域和TIR结构域,TRAF6存在4个保守结构域分别为RING结构域、2个锌指结构域、环-环(coiled-coil)结构域以及TRAF同源结构域(meprinand TRAF-homology, MATH),并与其他物种氨基酸序列一致性较高。系统进化树分析发现斑马鱼myd88和traf6基因与硬骨鱼类亲缘关系较近,说明斑马鱼myd88和traf6基因的同源性符合其在物种进化上的地位并且具有很高的结构同源性。将斑马鱼myd88和traf6基因的CDS全长序列连接至pCMV-Tag2B表达载体。通过对重组质粒进行双酶切检测发现,成功克隆了斑马鱼pCMV-myd88和pCMV-traf6真核表达载体。为了验证这2个真核表达载体的生物学功能,本研究在HEK293T细胞中进行NF-κB的报告基因活性检测。结果表明,过表达myd88和traf6基因后,斑马鱼NF-κB家族nfκb1启动子的转录活性显著升高,分别约为空载对照组的2.5倍和8倍。鉴于MYD88和TRAF6在天然免疫功能等方面的重要作用,实验结果为以后研究斑马鱼的先天免疫信号传导过程提供了有力的研究工具。Both myeloid differentiation primary response gene 88(MYD88) and TNF receptor associated factor 6(TRAF6) are key adaptor molecules in the Toll-like receptors family(TLR). Zebrafish is a unique model organism for studying the innate immune response. In order to construct eukaryotic expression vectors of myd88 and traf6,and use them to study the immune response mechanism, we cloned the full-length CDS sequence of the coding region of zebrafish myd88 and traf6 genes in this study. The full length of zebrafish myd88 and traf6 gene CDS was855 bp and 1 629 bp, respectively. Structural analysis showed that there were two conserved domains of zebrafish MYD88 including DD domain and TIR domain;four conserved domains of TRAF6 including RING domain, two zf domains, coiled-coil domain and MATH domain. And they had high amino acid sequence identity with other species. Phylogenetic tree analysis found that the zebrafish myd88 and traf6 gene had high structural homology and closely related to teleost fish, indicating that they were consistent with their evolutionary status. Then the CDS sequence of myd88 and traf6 genes were constructed to the expression vector of pCMV-Tag2 B. Zebrafish pCMV-myd88 and p CMV-traf6 eukaryotic expression vectors were successfully cloned by double enzyme digestion.In order to verify the biological function of these two eukaryotic expression vectors, we performed NF-κB reporter gene verification in HEK293 T cell. After overexpression of myd88 and traf6 gene, the transcriptional act ivity of nfκb1 in zebrafish NF-κB family was significantly increased, about 2.5 and 8 times, respectively, compared with that of the control group. In view of the important role of MYD88 and TRAF6 in innate immune function, our results provided a powerful research tool for the future study on the innate immune signal transduction process of zebrafish.

关 键 词：斑马鱼 pCMV-myd88 pCMV-traf6 真核表达载体 

分 类 号：Q78[生物学—分子生物学]