突触小泡膜蛋白VAMP-2的原核表达、纯化及活性鉴定

摘要

目的 克隆突触小泡膜蛋白(VAMP-2)基因,原核表达、纯化并鉴定重组蛋白VAMP-2活性.方法 根据GenBank中已报道的VAMP-2基因序列,设计特异性引物,通过RT-PCR从小鼠的全脑组织总RNA中获得基因.将去疏水区后的基因克隆至含有N端信号肽pelB序列的原核表达载体pET-22b中,重组质粒转化BL21(DE3) Rosetta感受态细胞,IPTG诱导表达,表达产物经Ni-NTA亲和层析纯化,SDS-PAGE及Western印迹进行鉴定,并利用金属内肽酶BoNT/B轻链对该蛋白进行生物活性分析和酶活动力学测定.结果 与结论成功构建pET-22b-VAMP-2表达质粒,并转化大肠杆菌BL21(DE3)Rosetta,Western印迹证实重组蛋白VAMP-2(残基Met 1-Met 94)获得可溶性表达.重组蛋白VAMP-2可被金属内肽酶BoNT/B特异性酶解,具有良好的生物学活性.%Objective To clone the vesicle-associated membrane protein ( VAMP)-2 gene and to express, purify and identify VAMP. Methods According to the sequence of VAMP-2 gene from GenBank , a DNA fragment encoding VAMP-2 was obtained from the mouse brain by RT-PCR and inserted into pET-22b to create plasmid pET-22b-VAMP-2. The recom-binant plasmid was transformed into BL21 (DE3) Rosetta and induced by IPTG at 20℃. VAMP-2 with His-tag was purified using Ni2+ -TA agarose. VAMP-2 cleft by light chain (LC) of BoNT/B was examined by SDS-PAGE. Results and Conclusion The expression plasmid pET22b-VAMP-2 was successfully constructed , transformed into the E. coll strain BL21 (DE3) Rosetta, and induced by IPTG to express the recombinant VAMP-2. After purification , VAMP-2 was analyzed by Western-blotting. The activity of the protein was also analyzed by a metallic endopeptidase BoNT /B. VAMP-2 gene has been successfully cloned and expressed ,and its product has been purified and identified .