马立克氏病毒UL36蛋白的表达及其去泛素化酶活性分析  

Expression of UL36 protein of Marek's disease virus and its activity of deubiquitinase

作　　者：王梦云[1] 周宏达[1] 韦美甜 吕岩[1] 吴山力[2] 艾永兴[1] 

机构地区：[1]吉林大学动物科学学院,吉林长春130062 [2]吉林大学白求恩医学院,吉林长春130021

出　　处：《畜牧与兽医》2017年第5期77-82,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金资助项目(31272528);教育部新世纪优秀人才支持计划资助项目(NCET-12-0232);教育部留学回国人员科研启动基金

摘　　要：选取马立克氏病毒(MDV)去泛素化酶UL36蛋白N端具有蛋白酶活性的一段氨基酸序列,表达纯化并分析UL36的去泛素化酶活性特点。利用p Cold TF为表达载体,并在UL36-323的C端连接Strep tagⅡ作为纯化标签,构建重组质粒p Cold TF-UL36-Strep。应用大肠杆菌表达体系,低温诱导表达,并对该段蛋白进行纯化,得到可溶且纯度较高的蛋白后,通过Western blot确定所纯化的蛋白即是UL36蛋白。应用Di-Ub(K48)和SUMO1-GST作为底物鉴定其去泛素化酶活性。最终得到了可溶且纯度较高的UL36-323-Strep蛋白,并且通过酶切反应确定了其去泛素化酶活性,为进一步探究UL36在MDV感染及复制中的作用奠定前期基础,也为探究MDV的致病机理提供了必要的前提条件。To characterize the deubiquitinating property of UL36 protein of Marek＇s disease virus （MDV）, a DNA fragment encoding 323 amino acids of UL36 N-terminus was cloned into pColdTF-C-StrepTag II expression plasmid and then expressed in E. coli BL21 （DE3） ceils at low temperature. UL36-323 protein fused with C-terminal Strep Tag H was purified on Streptactin column. The purified UL36-323 protein was determined by Western blot with primary antibody against UL36. The K48 linked ubiquitin dimer （Di-Ub （K48）） and SUMO1 -GST were used as substrates to identify deubiquitinating activity of UL36-323. The results showed that purified UL36-323 could specifically deconjugate ubiquitin substrate, Di-Ub （K48）, but not SUMO substrate, SUMO-GST. The data in this study lay a foundation for further study on the function of UL36 in MDV-indueed tumorigenesis and pathogenesis.

关 键 词：MDV UL36 蛋白纯化 去泛素化酶 

分 类 号：Q78[生物学—分子生物学]