Expression and identification of cysteine proteinase from adult worm of Pagumogonimus skrjabini in Escherichia coli
- VernacularTitle:斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因片段的表达及鉴定
- Author:
Wenyuan DUAN
;
Xilin ZHANG
;
Ying WANG
;
- Publication Type:Journal Article
- Keywords:
Pagumogonimus skrjabini;
cysteine protease;
fusion protein;
Western blotting;
immunoreactivity
- From:Journal of Third Military Medical University
2003;0(08):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express the recombinant antigen coded by Pagumogonimus skrjabini adult cysteine protease gene fragment and to investigate the immunoreactivity of the fusion protein for further usage on serodiagnosis of the fluke disease. Methods The target gene fragment was amplified by PCR. After purification with gel purification recoverykit, the target gene fragment was ligated with PinPointTM Xa 1 T vector and transduced into E.coli JM109 strain. The expressed fusion protein sample was prepared with alkaline lysis solution, and then analyzed by SDS PAGE. The expression level was determined by Coomassie blue staining and the streptavidin alkaline phosphatase staining. The immunoreactivity of fusion protein was examined by Western blotting. Results A total of 8 positive clones were harvested, and only one had the proper orientation verified by sequencing. The recombinant antigen was obtained after being induced with IPTG (Isopropltio ? D galactoside), and a positive band of 32?10 3 was found by streptavidin alkaline phosphatase staining. In the same position, the fusion protein was also detected by Western blotting. Conclusion The expression vector of adult cysteine protease gene PinPointTM Xa 1 T vector was successfully constructed. The recombinant antigen obtained after being induced with IPTG possesses good immunoreactivity.
