Cloning,expression and bioinformatics analysis of cathepsin B of Echino-coccusgranulosus
- VernacularTitle: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
- Author:
Ting ZHANG
;
Lifang JIA
;
Ying CHEN
;
Chuan JU
;
Xiaojin MO
;
Bin XU
;
Shenbo CHEN
;
Junhu CHEN
;
Wei HU
- Publication Type:Journal Article
- Keywords:
Echinococcus granulosus;
Cathepsin B;
In-Fusion clone;
Cell-free system;
Bioinformatics
- From:
Chinese Journal of Schistosomiasis Control
2014;(6):642-647
- CountryChina
- Language:Chinese
-
Abstract:
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.
