CAJ | 학술논문

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、Cys112、His282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。
목적：극륭、표체세립극구조충조직단백매B（EgCatB）기인，병대기진행생물신식학예측화분석。방법제취세립극구조충총RNA반전록성cDNA，이차위모판확증목적기인。이용무봉극륭기술화맥배무세포표체체계극륭표체EgCatB，용면역인적실험진행험증。채용SignalP 4.1、TMHMM sever v.2.0、TargetP 1.1대EgCatB편마단백분별진행신호태、과막구급아세포정위적예측。채용BLASTP화GeneDoc진행EgCatB동원서렬비대급보수위점분석。채용ProtParam、SMART、Predictprotein、Swiss?model분석EgCatB편마단백적결구。채용NetOGlyc 4.0 Server화NetNGlyc 1.0 Server재선분석EgCatB단백O형화N형당기화위점。결과성공구건료중조질립pEU?EgCatB。단백전영화면역인적실험결과현시해기인획득료가용성표체。경생물신식학분석예측해단백분자량35.9 kDa、이론등전점6.37，위함신호태적분비단백，매활성위점고도보수，병통과Gln106、Cys112、His282화Asn302형성료최화중심。EgCatB기인소편마단백안기산서렬중불존재N?당기화위점，단존재9개O?당기화위점。결론성공극륭표체료세립극구조충EgCatB기인병대기진행료교전면적생물신식학예측분석，위해단백적공능연구제공료삼고의거。
Objective To clone and express cathepsin B gene of Echinococcus granulosus(EgCatB)and analyze EgCatB protein by using bioinformatics tools and online databases. 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 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,Cys112,His282 and Asn302. It was found that there were nine O?glycosylation sites in the EgCatB sequence,but no N?glycosylation sites. 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 informa? tion for further functional study of the EgCatB protein.