Construction of prokaryotic expression system of Taenia solium 14-3-3.2 and its expression at the stages of Taenia solium adult and cysticercus
10.3760/cma.j.cn231583-20200616-00160
- VernacularTitle:猪带绦虫14-3-3.2原核表达系统的构建及其在猪带绦虫成虫和囊尾蚴中的表达
- Author:
Meichen LIU
1
;
Renhui OUYANG
;
Bo LUO
;
Biying ZHOU
Author Information
1. 遵义医科大学寄生虫学教研室，贵州遵义 563000
- Keywords:
Taenia solium;
Cysticercus;
Ts14-3-3.2 protein;
Expression
- From:
Chinese Journal of Endemiology
2021;40(6):435-440
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish the prokaryotic expression system of Taenia solium (Ts) 14-3-3.2, and observe the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus. Methods:Based on the Ts14-3-3.2 gene sequence obtained by the Department of Parasitology, Zunyi Medical University in the previous study, the whole gene was synthesized by PCR-based accurate synthesis (PAS) method. After double digestion with restriction enzymes Nde Ⅰ and Xba Ⅰ, the plasmid pCzn1 was ligated to construct a recombinant plasmid pCzn1-Ts14-3-3.2. Then it was transformed into Escherichia coli ArcticExpress competent cells to induce the expression of Ts14-3-3.2 protein. The expression products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie blue staining. The purified Ts14-3-3.2 recombinant protein was obtained by Ni-affinity chromatography. New Zealand rabbits were immunized with the recombinant protein to produce Ts14-3-3.2 polyclonal antibody. Western blotting was used to detect the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus. Results:The recombinant plasmid pCzn1-Ts14-3-3.2 was successfully constructed. After induced expression, Ts14-3-3.2 target protein bands appeared in the supernatant and precipitated at the relative molecular weight of about 29.31 × 10 3. The purified Ts14-3-3.2 recombinant protein with His label could be recognized by anti-His monoclonal antibody, and the Ts14-3-3.2 polyclonal antibody with titer of 1 ∶ 512 000 was obtained. Western blotting showed that Ts14-3-3.2 protein was expressed at the stages of Ts adult and cysticercus. Conclusions:The prokaryotic expression system of Ts14-3-3.2 is successfully established, and the Ts14-3-3.2 polyclonal antibody with relatively higher purity and titer is obtained. The Ts14-3-3.2 protein is expressed at the stages of Ts adult and cysticercus.
