基于蛋白质反式剪接在大肠杆菌中表达鲎C因子CES多肽  

Expression of Tachypleus tridentatus Factor C Recombinant CES Peptide Using Intein-mediated Protein Trans-splicing

作　　者：王影影[1] 齐兴梅[2] 刘涛[3] 祁静[3] 张春[3] 郭玲玲[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,苏州215123 [2]苏州大学生命科学学院,苏州215123 [3]苏州分子诊断和治疗技术重点实验室,中国科学院苏州生物医学工程技术研究所,苏州215163

出　　处：《基因组学与应用生物学》2013年第6期707-712,共6页Genomics and Applied Biology

基　　金：便携式细菌内毒素快速检测仪及检测试剂(ZXY2012011);苏州市分子诊断和治疗技术重点实验室(ZXJ2-012005);用rAAV位点特异整合载体建立一株治疗帕金森氏症的分泌神经生长因子(GDNF)的成人脑神经星状前体细胞的细胞株(30970880)共同资助

摘　　要：鲎C因子是鲎血细胞中一种对内毒素具有高亲和力的丝氨酸蛋白酶原,可替代鲎试剂用于内毒素检测。鲎C因子N端Cys-EGF-sushi1-sushi2(CES)片段中的sushi1区域是与内毒素结合的关键部位。本研究在sushi1结构域中的特定位点断开CES与内毒素结合的功能片段,然后分别和蛋白质内含肽Ssp DnaX的N端片段(IN)和C端片段(IC)连接,并在大肠杆菌中表达,表达产物经亲和层析纯化、复性以及内毒素去除后,利用蛋白质反式剪接系统,在体外诱导该蛋白质内含肽发生剪接,重新得到C因子的CES蛋白,并检测其活性。实验结果表明此重组CES蛋白具有结合内毒素活性,提示在大肠杆菌系统中开发低成本内毒素检测试剂的可行性。Factor C is a serine protease in Tachypleus tridentatus blood cells which can be highly-affiliated to endotoxins. It has been widely used in the detection of endotoxins. The sushi domain of CES in the N-terminal part is critical in combination with endotoxins. The recombinant CES protein can be synthesized in E. coli using intein-mediated protein trans-splicing. The N-terminal intein （IN） and C-terminal intein （IC） of a modified Ssp DnaX mini-intein were tagged with 6-His. CES-IN and CES-IC proteins were expressed in E. coli respectively. After His-select affinity chromatography and removal of endotoxin, the purified endotoxin-free fusion proteins were mixed for a trans-splicing reaction releasing Tachypleus tridentcaus factor C recombinant CES protein. The function of the recombinant CES was characterized by neutralization of endotoxin. The results showed that this recombinant CES protein could be successfully combined with endotoxins, indicating that it could be used in the development of Limulus/Tachypleus Amebocyte Lysate at a low cost.

关 键 词：反式剪接 内含肽 东方鲎C因子 CES多肽 内毒素 荧光检测 

分 类 号：Q953[生物学—动物学]