突触小泡膜蛋白VAMP-2的原核表达、纯化及活性鉴定  

Prokaryotic expression,purification and identification of VAMP-2

作　　者：罗森[1,2] 王琴[2] 房华丽[2] 王德慧[2] 李崭[2] 李涛[2] 王慧[2] 

机构地区：[1]安徽医科大学,合肥230032 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2013年第4期250-253,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31201352)

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 recombinant plasmid was transformed into BL21（DE3） Rosetta and induced by IPTG at 20℃.VAMP-2 with His-tag was purified using Ni2＋-NTA 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.coli 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.

关 键 词：囊泡相关膜蛋白质2 金属内肽酶 底物 原核表达 

分 类 号：Q78[生物学—分子生物学]