Expression and purification of soluble fusion protein of Toxoplasma gondii GRA6 in E.coli
- VernacularTitle:弓形虫GRA6可溶性蛋白在大肠埃希菌中的表达及纯化
- Author:
Xiang ZHU
;
Tai-Shan MIN
;
Shenglan GAO
;
Al ET
;
- Publication Type:Journal Article
- Keywords:
Toxoplasma;
Cytoplasmic granules;
Antigens,protozoan;
Recombinant fusion proteins;
Gene expression
- From:
Chinese Journal of Infectious Diseases
2001;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To high express and purify toxoplasma gondii antigen GRA6 in E.coli which can be used to develop the genetic engineering diagnostic reagents.Methods The recombinant plasmid of pGEX-GRA6 was transformed to a bacterium BL21-Codon Plus(DE3)-RP and the recom- binant product was expressed under the inducement of isopropyl-beta-D-thiogalactosidase(IPTG). Cells were lysed by multiple rounds of sonication.The expression product was analyzed by using SDS- PAGE.Furthermore,it was purified by sedimentation of ammonium sulphate,desalting using Sephe- dax GS0 and affinity chromatography on glutathione-sepharose system.The immunogenicity of recom binant antigen purified was tested with Western blot.Results GRA6 was highly expressed in E.coli as fusion protein consisting of glutathione S-transferase and GRA6(GST-GRA6).The solubility anal- ysis of expression product indicated that this recombinant protein could be expressed in both superna- tant and inclusion bodies.The soluble protein of GRA6 in supernatant yield the final preparation at greater than 90% after purification.The recombinant protein purified could be recognized by human toxoplasmosis-infective sera with Western blotting analysis.Conclusions The soluble protein of GRA6 was highly expressed in E.coli and the recombinant antigen could be recognized by human tox- oplasmosis-infective serum after purification.The recombinant antigen can be used for devoloping kit to diagnose the acute and chronic infection of toxoplasma gondii.
