突触小体相关蛋白SNAP25的原核表达、纯化及鉴定

目的:克隆突触小体相关蛋白(SNAP25)基因,原核表达、纯化并鉴定SNAP25蛋白。方法:PCR扩增SNAP25基因,克隆至表达质粒pTIG-Trx,转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达,Ni2+-NTA亲和层析纯化目的蛋白,SDS-PAGE及Western印迹分析肉毒神经毒素BoNT/A轻链对该蛋白的裂解情况。结果:构建了pTIG-SNAP25表达质粒,经IPTG诱导表达,目的蛋白占全菌蛋白的26.2%,表达形式为可溶性表达,表达量达115.4mg/L,纯化后蛋白纯度达95%以上;经SDS-PAGE及Western印迹分析,SNAP25蛋白可被BoNT/A轻链特异降解。结论:克隆了SNAP25基因,在原核系统中表达、纯化并鉴定了重组SNAP25蛋白。

Prokaryotic Expression and Purification of SNAP25 and its Identification

Objective：To clone,express,purify and identify the synaptosomal associated protein of molecular mass 25kD（SNAP25） in Escherichia coli.Methods：The SNAP25 gene was generated by PCR method and inserted into pTIG-Trx to create plasmid pTIG-SNAP25.The recombinant plasmid was transformed into the E.coli strain BL21（DE3） and induced by IPTG at 25℃.SNAP25 protein with His-tag was purified using Ni2＋-NTA agarose.The SNAP25 being cleavaged by light chain（LC） of BoNT/A was examined by SDS-PAGE and Western blot.Results：The expression plasmid pTIG-SNAP25 was successfully constructed.The protein expressed accounted for 26.2% of total bacterial protein with a yield as high as 115.4 mg/L.Its purity was up to 95% after purification.The results of SDS-PAGE and Western blot showed that SNAP25 protein could be cleaved by BoNT/A LC protein.Conclusion：SNAP25 gene has been successfully cloned and expressed,with its product has been purified and identified.