乳酸乳球菌肽聚糖锚钩蛋白原核表达载体的构建与表达

Construction of prokaryotic expression vector of Lactococcus lactis peptidoglycan protein anchor and its expression

以乳酸乳球菌肽聚糖锚钩蛋白(Protein anchor,PA)为研究对象,构建原核表达载体并进行表达,以期获得PA融合蛋白。首先应用PCR技术扩增得到乳酸乳球菌PA基因片段;然后应用pET-32a(+)质粒构建原核表达载体,将其转化大肠杆菌BL-21(DE3),进行诱导表达,最后进行融合蛋白的纯化及锚定活性鉴定。结果显示:PA融合蛋白在大肠杆菌中获得表达,主要以包涵体形式存在;包涵体经纯化复性后的PA蛋白可与肽聚糖结合。说明乳酸乳球菌PA蛋白在大肠杆菌中可高效表达并具有锚定活性,为今后该蛋白多克隆抗体制备及与目的抗原的融合表达等相关研究奠定了基础。

This study aimed to construct the prokaryotic expression vector of Lactococcus lactis peptidoglycan protein anchor （PA） and to obtain PA fusion protein. The PA gene sequence amplified by PCR amplification was ligated with pET- 32a （＋） to construct prokaryotic expression vector. The vector was transformed into Escherichia coli Bl221 （ DE3 ） and was induced to express fusion protein. PA fusion protein was expressed successfully in the form of inclusion bodies. The puri- fied and refolded PA protein could bind to L. lactis peptidoglycan. These provided a foundation for preparation of PA poly- clone antibody and the fusion expression with target antigen.