Chin J Schisto Control ›› 2014, Vol. 26 ›› Issue (6): 642-.

Previous Articles     Next Articles

Cloning, expression and bioinformatics analysis of cathepsin B of Echinococcus granulosus

ZHANG Ting1 |JIA Li-fang 1 |CHEN Ying1| 2 |JU Chuan1 |MO Xiao-jin1 | XU Bin1* |CHEN Shen-bo1 |CHEN Jun-hu1 | HU Wei 1   

  1. 1 National Institute of Parasitic Diseases|Chinese Center for Disease Control and Prevention;Key Laboratory of Parasite and Vec? tor Biology|Ministry of Public Health;WHO Collaborating Centre for Malaria|Schistosomiasis and Filariasis|Shanghai 200025|China; 2 College of Traditional Mongolian Medicine| Inner Mongolia University for the Nationalities|China
  • Online:2014-12-22 Published:2014-12-23
  • Contact: XU Bin

细粒棘球绦虫组织蛋白酶B的重组表达及生物信息学分析

张颋1|贾利芳1|陈英1,2|鞠川1|莫筱瑾1|徐斌1*|陈绅波1|陈军虎1|胡薇1   

  1. 1 中国疾病预防控制中心寄生虫病预防控制所| 卫生部寄生虫病原与媒介生物学重点实验室| 世界卫生组织疟疾、 血吸虫病和丝虫 病合作中心 (上海 200025); 2 内蒙古民族大学蒙医药学院
  • 通讯作者: 徐斌
  • 作者简介:张颋| 女| 博士| 助理研究员。研究方向: 寄生虫分子生物学
  • 基金资助:
    国家自然科学基金(81201315); 中国疾病预防控制中心青年基金 (2013A103); 上海市卫生局科研课题 (20124174); 上海市自然科 学基金 (13ZR1462800)

Abstract: Objective Objective To clone and express cathepsin B gene of Echinococcus granulosus(EgCatB)and analyze EgCatB protein by using bioinformatics tools and online databases. Methods Methods The total RNA of E. granulosus was extracted and reverse? ly transcribed into cDNA as the template sequence for PCR. The EgCatB gene was cloned by using the In?Fusion PCR cloning method and expressed by a wheat germ cell?free system,and then the recombinant protein was identified by Western blotting. The signal peptide,transmembrane helices and subcellular location of the EgCatB sequence were predicted by the online soft? ware SignalP 4.1,TMHMM sever v. 2.0 and TargetP 1.1 respectively. Subsequently,the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc software. Finally,the structures and the glycosylation modification site of the EgCatB encoding protein were analyzed and predicted in turn by ProtParam,SMART,Predictprotein,Swiss?model,NetOGlyc 4.0 and NetNGlyc 1.0 approaches. Results Results The EgCatB gene was successfully amplified from cDNA of E. granulosus and ex? pressed in the soluble fractions. The molecular weight of the expressed protein was estimated 35 kDa. The bioinformatics analysis revealed that EgCatB was a classical secreted protein containing a Pept_C1 domain. The homology analysis indicated that the amino acid sequence of EgCatB was highly conserved in the active enzyme sites. The protein structure prediction showed a cata? lytic active center was formed through Gln106 ,Cys 112 ,His 282 and Asn302 . It was found that there were nine O?glycosylation sites in the EgCatB sequence,but no N?glycosylation sites. Conclusions Conclusions The EgCatB gene is cloned and expressed successfully,and the recombinant protein is analyzed by bioinformatics approaches and structure predication. The study provides useful information for further functional study of the EgCatB protein.

Key words: Echinococcus granulosus;Cathepsin B;In?Fusion clone;Cell?free system; Bioinformatics

摘要: 目的 目的 克隆、 表达细粒棘球绦虫组织蛋白酶B (EgCatB) 基因, 并对其进行生物信息学预测和分析。方法 方法 提取 细粒棘球绦虫总RNA反转录成cDNA, 以此为模板扩增目的基因。利用无缝克隆技术和麦胚无细胞表达体系克隆表达 EgCatB, 用免疫印迹实验进行验证。采用SignalP 4.1、 TMHMM sever v. 2.0、 TargetP 1.1对EgCatB编码蛋白分别进行信号 肽、 跨膜区及亚细胞定位的预测。采用BLASTP和GeneDoc进行EgCatB同源序列比对及保守位点分析。采用ProtParam、 SMART、 Predictprotein、 Swiss?model分析EgCatB编码蛋白的结构。采用NetOGlyc 4.0 Server和NetNGlyc 1.0 Server在线分 析EgCatB蛋白O型和N型糖基化位点。结果 结果 成功构建了重组质粒pEU?EgCatB。蛋白电泳和免疫印迹实验结果显示 该基因获得了可溶性表达。经生物信息学分析预测该蛋白分子量35.9 kDa、 理论等电点6.37, 为含信号肽的分泌蛋白, 酶 活性位点高度保守, 并通过Gln106 、 Cys 112 、 His 282 和Asn302 形成了催化中心。EgCatB基因所编码蛋白氨基酸序列中不存在N? 糖基化位点, 但存在9个O?糖基化位点。结论 结论 成功克隆表达了细粒棘球绦虫EgCatB基因并对其进行了较全面的生物 信息学预测分析, 为该蛋白的功能研究提供了参考依据。

关键词: 细粒棘球绦虫; 组织蛋白酶B; 无缝克隆; 无细胞体系; 生物信息学

CLC Number: