SNAP-25基因克隆及其在大肠杆菌中的表达与鉴定  

Cloning,expression and identification of SNAP-25 gene in Escherichia coli

作　　者：刘昊[1] 史晶[1] 蔡昆[1] 高翔[1] 侯晓军[1] 王慧[1] 

机构地区：[1]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：《军事医学科学院院刊》2009年第3期228-230,共3页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:构建SNAP-25基因的原核表达载体pET22b-SNAP-25,对表达产物活性进行初步鉴定。方法:根据GenBank中已报道的SNAP-25基因序列,设计特异引物,通过RT-PCR从小鼠全脑组织总RNA中得到基因。将全长基因克隆至原核表达载体pET22b中,重组质粒转化大肠杆菌E.coliBL21感受态细胞,IPTG诱导表达,表达产物经Ni-NTA亲和层析进行纯化。通过SDS-PAGE和免疫印迹对其进行鉴定,并对该蛋白进行活性的初步分析。结果与结论:成功构建了原核表达载体pET22b-SNAP-25,Western印迹证实重组蛋白获得表达。表达的重组蛋白与A型肉毒毒素混合反应,经SDS-PAGE验证,重组蛋白具有被毒素特异性切割的活性。Objective：To construct a prokaryotic expression vector pET22b-SNAP-25 of SNAP-25 gene, and identify biological activity of SNAP-25 protein. Methods：According to the sequence of SNAP-25 gene from GenBank, a DNA fragment encoding SNAP-25 was obtained from the mouse brain by RT-PCR, and the product was connected with pMD-18T and transformed into DH5α. After DNA sequencing, the positive clones were picked out. The cleaved SNAP-25 fragment was cloned into prokaryotic vector pET22b. The recombinant plasmid pET22b-SNAP-25 was transformed into E. coli BL21, and induced by IPTG to express the recombinant SNAP-25. After purification, the SNAP-25 was analyzed by Western-blot. The activity of the protein was also analyzed. Results and Conclusion：There was a new band of protein around 28 × 10^3 on SDS-PAGE,which was proved by Western-blot to be SNAP-25, and the recombinant SNAP-25 could be cleaved by botulinum neurotoxin （BoNT）.

关 键 词：SNAP-25 肉毒毒素 底物 原核表达 

分 类 号：Q78[生物学—分子生物学]