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

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）.