虾肝肠胞虫类泛素蛋白EhUBL的原核表达与亚细胞定位  

Prokaryotic Expression and Subcellular Localization of the Ubiquitin-Like Protein EhUBL from Enterocytozoon hepatopenaei

作　　者：谭念秋 魏春梅 文钰凇 范晓东[1,2] 周泽扬[1,2] TAN Nianqiu;WEI Chunmei;WEN Yusong;FAN Xiaodong;ZHOU Zeyang(Chongqing Key Laboratory of Animal Biology,School of Life Sciences,Chongqing Normal University,Chongqing 401331;Chongqing Key Laboratory of Microsporidium Infection and Control,Southwest University,Chongqing 400715,China)

机构地区：[1]重庆师范大学生命科学学院、动物生物学重庆市重点实验室,重庆401331 [2]西南大学微孢子虫感染与防控重庆市重点实验室,重庆400715

出　　处：《重庆师范大学学报(自然科学版)》2024年第6期43-53,共11页Journal of Chongqing Normal University:Natural Science

基　　金：国家自然科学基金面上项目(No.32372944);重庆市技术创新与应用示范项目(No.cstc2018jscx-msybX0313);重庆市教育委员会科学技术研究计划青年项目(No.KJQN201900516);重庆市教育委员会中小学创新人才培养工程项目(No.CY230503);重庆师范大学基金项目(No.22XLB023)。

摘　　要：为明确虾肝肠胞虫(Enterocytozoon hepatopenaei)类泛素蛋白家族成员EhUBL的序列特征和定位情况,利用Expasy、WegoLoc、Interpro等在线软件对EhUBL的序列特征和空间结构进行预测分析,通过对它的编码基因EhUBL进行克隆、原核表达以获得EhUBL重组蛋白(rEhUBL),并制备特异性多克隆抗体anti-EhUBL,再通过免疫电镜探查EhUBL的亚细胞定位情况。结果显示:1)EhUBL全长873 bp,编码292个氨基酸;EhUBL的预测相对分子质量为31.38 kDa,该蛋白分子拥有UBL(ubiqutin-like)-UBA(ubiquitin associated)结构域。2)EhUBL与黄道蟹肠胞虫(Enterospora canceri)假定蛋白ECANGB1_154和毕氏肠胞虫(Enterocytozoon bieneusi)假定蛋白EBI_25559的亲缘关系较近。3)通过基因克隆和原核表达成功获得了rEhUBL,并制备了anti-EhUBL。4)EhUBL主要分布在虾肝肠胞虫孢子的胞壁及细胞核中。上述结果为EhUBL的功能研究奠定基础,为虾肝肠胞虫的防控提供了新靶标。In order to clarify the sequence characterisation and localisation analysis of the ubiquitin-like protein in Enterocytozoon hepatopenaei(EhUBL),the sequence features and spatial structure of EhUBL were predicted and analysed using online websites such as Expasy,WegoLoc and Interpro,the recombinant EhUBL(rEhUBL)was obtained by cloning its coding gene EhUBL and prokaryotic expression and the specific polyclonal antibody(anti-EhUBL)was prepared,and then the subcellular localisation of EhUBL was probed by immunoelectron microscopy.The results showed that:1)The length of EhUBL was 873 bp,it encoded 292 amino acids;the predicted relative molecular mass of EhUBL was 31.38 kDa,and the protein had the UBL(Ubiqutin-like)-UBA(Ubiquitin associated)domain.2)EhUBL was closely related to the hypothetical protein ECANGB1_154 of Enterospora canceri and the hypothetical protein EBI_25559 of Enterocytozoon bieneusi.3)rEhUBL was successfully obtained by gene cloning and prokaryotic expression,and anti-EhUBL was also successfully prepared.4)EhUBL was mainly distributed in the cell wall and nucleus of the spore of E.hepatopenaei.The results above laid a foundation for the functionl study of EhUBL and provided a new target for the prevention and control of E.hepatopenaei.

关 键 词：虾肝肠胞虫 泛素化 UBL(ubiqutin-like)-UBA(ubiquitin associated)结构域 原核表达 亚细胞定位 

分 类 号：Q786[生物学—分子生物学] S917.1[农业科学—水产科学]