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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。方法提取细粒棘球绦虫Total-RNA并反转录成c DNA,以此为模板扩增目的基因。将目的基因连接至p ET28b构建重组质粒并转化大肠杆菌BL21(DE3)进行重组表达。采用Signal P4.1、TMHMM sever v.2.0和Target P1.1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Gene Doc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。结果成功克隆并构建重组质粒p ET28bEgPDH,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。
Objective To clone and express the gene of pyruvate dehydrogenase (EgPDH) of Echinococcus granulosus and predict its bioinformatics. Methods Total RNA of Echinococcus granulosus was extracted and reverse transcribed into c DNA to amplify the target gene. The target gene was ligated to p ET28b to construct a recombinant plasmid and transformed into E. coli BL21 (DE3) for recombinant expression. SignalP1, TMHMM sever v.2.0 and Target P1.1 were used to predict the signal peptide, transmembrane region and subcellular localization of EgPDH-encoded protein sequences respectively. The protein domain of EgPDH was screened by SMART. The homologous sequences of EgPDH and conserved sites were analyzed by BLASTP and Gene Doc. The phylogenetic tree was constructed by MEGA6 software. Results The recombinant plasmid p ET28bEgPDH was successfully cloned and constructed. The target gene was about 1 080 bp in size and the recombinant protein was expressed in soluble form. EgPDH is a secreted protein of the signal peptide and contains a transketolase domain. The highly conserved enzyme sites of the protein are Glu57, Leu72, Ile86 and Phe114. Phylogenetic tree analysis showed that EgPDH had the closest genetic relationship with PDH of Echinococcus multilocularis. Conclusion The EgPDH gene of Echinococcus granulosus was cloned successfully and predicted by bioinformatics analysis, which laid the foundation for further study on the function of this protein.