高可溶性Aβ融合蛋白的表达、纯化和鉴定

Expression, purification and identification of Aβ fusion protein with enhanced solubility

目的在大肠杆菌中表达阿尔茨海默病(AD)相关肽段Aβ与具有强助溶作用的麦芽糖结合蛋白(MBP)的融合蛋白,从而经济有效地获得具有天然免疫原性的Aβ肽段,为深入研究AD的发病机理和治疗药物提供重要的物质基础。方法用PCR法扩增Aβ的基因片段,并将其插入到含有麦芽糖结合蛋白的融合表达载体pMAL-c2中,经测序后再将重组质粒转化入大肠杆菌TB1中进行诱导表达。经多聚麦芽糖亲和色谱的纯化获得MBP-Aβ融合蛋白。再用SDS-PAGE及Western blotting鉴定Aβ及其融合蛋白的表达和免疫原性。结果测序结果证实插入的DNA片段与Aβ序列一致。SDS-PAGE电泳显示Aβ融合蛋白被高效表达。Western blotting结果表明Aβ及其融合蛋白可被其特异性抗体识别。结论在大肠杆菌中高效表达了可溶性的Aβ融合蛋白并经纯化和酶解后获得了Aβ纯肽段,为AD病机理的研究及其病理模型的建立提供了物质基础。

Objective To economically obtain the Aβ peptide for Alzheimer＇s disease （AD） research by expressing the Aβ peptide fused with the maltose binding protein （MBP） possessing high solubiliry in E.coli. Methods The cDNA-coding sequence of Aβ peptide was modified by the addition of a BamH I site at the 5＇end and a Hind III site at the 3＇ end using PCR. The modified sequence was ligated into the maltose-binding protein （MBP） fusion expression vector pMAL-c2 containing an thrombin cleavage site, which was transformed into competent E.coli DH5α cells. After identification of the single clones by PCR and DNA sequencing, the recombinant plasmid was transformed into E.coli TB1 and induced to express MBP-Aβ fusion protein. The expressed fusion protein was purified using amylose resin column and identified by SDS-PAGE and Western blotting. Results The result of DNA sequencing verified the consistency between the inserted sequence and Aβ （1-42） sequence. SDS-PAGE electrophoresis showed that MBP-Aβ fusion protein was highly expressed in E.coli TB1, and Western blotting demonstrated that the purified fusion protein and the separated Aβ peptide could be recognized by specific anti-Aβ （22-35） antibody. Conclusion MBP-Aβ fusion protein highly expressed in E. coli TB1 cells with enhanced solubility and the separated Aβ peptide with good immunogenicity obtained may lend support to AD research.