Cloning, expression, purification and identification of EgG1Y162-2 gene from Echinococcus granulosus
10.3760/cma.j.cn231583-20201226-00344
- VernacularTitle:细粒棘球蚴EgG1Y162-2基因的克隆、蛋白表达及纯化鉴定
- Author:
Huifang KONG
1
;
Shangqi ZHAO
;
Yanxia ZHOU
;
Qiaoqiao GONG
;
Yujiao LI
;
Chunbao CAO
;
Haimei MA
;
Jianbing DING
;
Xiaotao ZHOU
Author Information
1. 新疆医科大学基础医学院免疫学教研室，乌鲁木齐 830011
- Keywords:
Echinococcus granulosus;
EgG1Y162-2 protein;
Expression;
Purification
- From:
Chinese Journal of Endemiology
2021;40(8):635-639
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct the pET30a-EgG1Y162-2 prokaryotic expression plasmid and induce the expression of EgG1Y162-2 protein, so as to provide a research basis for development of Echinococcus granulosus vaccine. Methods:Using Echinococcus granulosus cDNA as a template, the target gene of EgG1Y162-2 was synthesized by PCR, and after digestion with restriction enzymes EcoRⅠ and Hind Ⅲ, it was connected to the prokaryotic expression vector pET30a to construct the recombinant plasmid pET30a-EgG1Y162-2. The recombinant plasmid was transformed into competent cell BL21 (DE3) and induced by isopropyl β-D-thiogalactoside (IPTG) to express a large number of proteins. The recombinant protein was purified by affinity chromatography. The purification level was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression product was identified by Western blotting. Results:The recombinant plasmid pET30a-EgG1Y162-2 was successfully constructed. After inducting expression, the bacterial supernatant and the eluate were both at a relative molecular weight of about 15 × 10 3, and the protein antigen component eluted with 200 mmol/L imidazole was relatively pure. Western blotting results showed that the purified recombinant protein EgG1Y162-2 with His tag could be recognized by His monoclonal antibody. Conclusion:The pET30a-EgG1Y162-2 prokaryotic expression plasmid of Echinococcus granulosus is successfully constructed, and the recombinant protein of EgG1Y162-2 is induced to express, laying a foundation for further study on anti- Echinococcus granulosus vaccine.
