弓形虫RH株膜蛋白P30的原核表达与鉴定

Expression and identification of P30 surface antigen from RH strain of Toxoplasma gondii

目的在E.coli中表达弓形虫RH株膜蛋白P30。方法将P30基因定向克隆到pET28b,构建含目的基因的pET28b-P30重组质粒,转化E.coliBL21(DE3),接种含pET28b-P30的BL21(DE3)单菌落于LB培养基,1∶100稀释后用0.2mmol/L的IPTG诱导表达,SDS-PAGE和Westernblot鉴定诱导表达产物。结果1构建了重组质粒pET28b-P30,2在E.coli中表达了一分子量约为30kDa的融合蛋白,经Westernblot鉴定正确。结论在E.coli中高效表达了弓形虫RH株膜蛋白P30,并以包涵体形式存在。

Objective To express P30 surface antigen of RH strain of Toxoplasraa gondii in E. coli BL21 （DE3）. Methods The P30 gene from Toxoplasraa gondii was cloned to the pET28b vector after PCR, and the recombinant expression plasmid pET28b-P30 was constructed. Then the recombinants were transformed into E. coli BL21 （DE3） after identified by the restriction enzyme digestion, PCR and DNA sequence determination annlysis. A single colony of E. coli BL21 （DE3） containing the recombinant plasmid, pET28b-P30 was inoculated in LB culture, then diluted 1： 100 into 2 ml LB culture and induced by 0.2 mmol/L IPTG, and the expression product was identified by SDS-PAGE and Western blot. Results ① The recombinant plasmid of pET28b-P30 was constructed. ② Plasmid pET28b-P30 could express a specific 30 kDa fusion protein in E. coli BL21 （DE3）. Conclusions The expression plasmid which contains the gene fragment encoding P30 surface antigen of Toxoplasma gondii has been successfully constructed and is highly expressed in E. coli BL21 （DE3） as an inclusion body.