细粒棘球绦虫丙酮酸脱氢酶的重组表达及生物信息学分析

发布时间：2018-03-22 05:20

本文选题：细粒棘球绦虫　切入点：丙酮酸脱氢酶　出处：《中国血吸虫病防治杂志》2015年04期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to clone and express Echinococcus granulosus pyruvate dehydrogenase EgPDH gene. Methods Echinococcus granulosus Total-RNA was extracted from Echinococcus granulosus and transformed into c DNA, which was used as template to amplify the target gene. The target gene was ligated to p ET28b to construct recombinant plasmid and transformed into Escherichia coli BL21DE3. Signal P4.1TMHMM sever v.2.0 and Target P1.1 were used to signal the sequence of EgPDH encoding protein, respectively. The prediction of transmembrane region and subcellular localization. SMART was used to analyze the EgPDH coding protein domain, and BLASTP and Gene Doc were used to analyze the EgPDH homologous sequence alignment and conserved sites. The phylogenetic tree was constructed by MEGA6 software adjacency method. Results the recombinant plasmid pET28bEgPDHwas successfully cloned and constructed. The target gene size was about 1080bp. the recombinant protein expressed in soluble form was expressed as secretory protein of signal peptide and contained transketozyme domain. The highly conserved enzyme activity loci were Glu57 Leu72Ile86Phe114.The phylogenetic tree analysis showed that EgPDH was closely related to PDH of Echinococcus multilocularis. Conclusion the EgPDH gene of Echinococcus granulosus was cloned and analyzed by bioinformatics. It lays a foundation for further study of the function of the protein.
【作者单位】：内蒙古民族大学蒙医药学院;中国疾病预防控制中心寄生虫病预防控制所世界卫生组织疟疾、血吸虫病和丝虫病合作中心卫生部寄生虫病原与媒介生物学重点实验室;
【基金】：国家自然科学基金(81201315) 国家科技重大专项(2012ZX10004-220) 卫生部寄生虫与媒介生物学重点实验室开放课题(WSB-KTKT201304)
【分类号】：R383.3

【参考文献】