Expression and identification of the multiple gene ROP2-P30 of Toxoplasma gondii in E.coli BL21

VernacularTitle:刚地弓形虫ROP2-P30 复合基因在E.coli BL21中的表达和鉴定
Author: Dianbo ZHANG ; Qingkuan WEI ; Defu ZAI ; Yong CUI ; Jin LI ; Honggang GAO ; Xuelian BAI ; Lijiang ZHAO ; Guangdong HAN ; Keyi LIU
Publication Type:Journal Article
Keywords: T. gondii; P30 and ROP2 genes; fusion protein; western blot
From: Chinese Journal of Zoonoses 2006;(6):538-543
CountryChina
Language:Chinese
Abstract: To obtain the functional fusion protein of rhoptry protein 2, compound rhoptry protein2 and surface antigen 1 of Toxoplasma gondii. the ROP2 and P30 genes from genomic DNA of T.gondii RH strain were amplified by PCR, and were inserted into pMD18-T cloning vector. Then the ROP2 fragment was subcloned to pET-30a(+) plasmid digested by EcoRⅠand Hind Ⅲ to construct plasmid pET-ROP2. Furthermore,the P30 fragment was subcloned into pET-ROP2 digested by BglⅡand EcoRⅠto create plasmid pET-ROP2-P30, the resulting recombinant plasmids , transformed into E.coli BL21 (DE3), were induced with IPTG. and the proteins identified by SDS-PAGE were further purified and refolded. The biological activity was analyzed by Western blot with specific antibody. It was found that the sizes of ROP2 and ROP2-P30 were 1212 and 1896bp with corresponding molecular weight 50- kDa and 75-kDa, respectively. The recombinant protein ROP2 (50-kDa) could specifically react with rabbit-polyclonal antiserum, and complex fusion protein ROP2-P30 (75- kDa) could react with P30 monoclonal antibody.