Bioinformatic analysis on the Rap2B-like gene from Clonorchis sinnensis and its cloning and expression
10.3969/j.issn.1002-2694.2009.12.016
- VernacularTitle:华支睾吸虫Rap2B样基因的生物信息学分析及其克隆、表达
- Author:
Lexun WANG
;
Xinbing YU
;
Can HUANG
;
Ling LIU
;
Xuchu HU
;
Jin XU
- Publication Type:Journal Article
- Keywords:
Clonorchis sinensis;
Ras oncogene;
bioinformatics;
molecular cloning;
expression;
purification
- From:
Chinese Journal of Zoonoses
2009;(12):1195-1198
- CountryChina
- Language:Chinese
-
Abstract:
To analyze the structural characteristics of the Rap2B gene from Clonorchis sinensis by bioinformatics and to clone and express this gene through genetic engineering methods in order to obtain the corresponding recombinant protein.The structural and functional characteristics of the Rap2B protein were analyzed by using the related bioinformatic softwares. Using the full-length cDNA plasmid clone (Noc009e03) as template , the coding region of Rap2B gene sequence was amplified with PCR and cloned into the prokaryotic expression vector pET-28a(+) and then expressed in E.coli BL21 through IPIG induction. The recombinant protein expressed was purified by Ni-IDA affinity chromatography and detected by SDS-PAGE and Western blotting. It was demonstrated that the size of this gene was 847 bp in length, encoding 141 amino acids and its theoretical molecular weight was 15851.1 daltons. As demonstrated by PCR, double enzyme digestion and DNA sequencing, the recombinant expression plasmid pET-28a(+)-Rap2B was successively constructed and the Rap2B-like gene was highly expressed in E.coli BL21. The recombinant protein was obtained through purification with His affinity chromatography and could react specifically with the serum of rats infected with C.sinensis. Through these investigations, the Rap2B protein may be predicted as a member of the Ras oncogene family protein and is potential in the carcinogenic process of C.sinensis.
