Abstract:

Objective Objective To clone and express Echinococcus granulosus pyruvate dehydrogenase（EgPDH）gene and analyze EgPDH protein with bioinformatics tools and online database. Methods Methods The total RNAs of E. granulosus was extracted and re? versely transcribed into cDNA. The EgPDH gene was cloned into pET28b to construct the recombinant vector and expressed in E. coli BL21（DE3）system subsequently. The signal peptide，transmembrane helices and subcellular location in EgPDH se? quence were analyzed by the online software SignalP4.1，TMHMM sever v.2.0 and TargetP1.1，respectively. Subsequently，the structure of EgPDH was predicted by SMART. Finally，the homologue sequence and conserved sites were aligned by using BLASTP and GeneDoc among the homologous sequences of EgPDH. Based on the alignment of PDH sequence，an evolutionary tree of E. granulosus and other species were constructed by the neighbor joining method of MEGA6 software. Results Results The EgP? DH gene was successfully amplified from cDNA of E. granulosus and expressed in the soluble fractions. The bioinformatics anal? ysis revealed that EgPDH was a classical secreted protein and contained transketolase domain. The homology analysis revealed that the amino acid sequence of EgPDH was highly conserved in catalytic sites Glu57 ，Leu72 ，Ile86 and Phe114 . The phylogenetic tree analysis of PDH proteins showed the closest relationship between E. granulosus and E. multilocularis. Conclusion Conclusion An Eg? PDH gene is cloned and expressed successfully，and the recombinant protein is analyzed by the bioinformatics approaches and structure predication. The study provides useful information for further functional study of the EgPDH protein.

Key words: Echinococcus granulosus；Pyruvate dehydrogenase； Cloning；Expression； Bioinformatics

摘要：

目的 目的 克隆表达细粒棘球绦虫丙酮酸脱氢酶 （EgPDH） 基因， 并对其进行生物信息学预测与分析。 方法 方法 提取细粒棘球绦虫Total?RNA并反转录成cDNA， 以此为模板扩增目的基因。将目的基因连接至pET28b构建重组质粒并转化大肠杆菌BL21 （DE3） 进行重组表达。采用SignalP4.1、 TMHMM sever v.2.0和TargetP1.1对EgPDH编码蛋白序列分别进行信号肽、 跨膜区及亚细胞定位的预测。采用SMART分析EgPDH编码蛋白结构域， 用BLASTP和GeneDoc进行EgPDH同源序列比对及保守位点分析， 并采用MEGA6软件邻接法构建系统进化树。 结果 结果 成功克隆并构建重组质粒pET28b? EgPDH， 目的基因大小约1 080 bp， 重组蛋白以可溶性形式表达。EgPDH为信号肽的分泌蛋白， 并含转酮酶结构域， 其高度保守酶活性位点分别为Glu57 、 Leu72 、 Ile86 、 Phe114 。系统进化树分析显示EgPDH与多房棘球绦虫PDH亲缘关系最近。 结 结论 论 成功克隆表达了细粒棘球绦虫EgPDH基因并进行了生物信息学预测分析， 为进一步研究该蛋白功能奠定了基础。

关键词: 细粒棘球蚴绦虫； 丙酮酸脱氢酶； 克隆； 表达； 生物信息学