人P53的原核表达及包涵体复性研究

通过检测血清中p53蛋白及其抗体变化诊断肿瘤的ELISA试剂盒的研发需要大量制备p53蛋白.本研究通过构建人野生型p53基因的原核表达质粒pET-32a-p53,在大肠杆菌中诱导表达得到p53包涵体蛋白,经包涵体变性、亲和纯化、复性得到可溶p53蛋白.发现利用8M尿素对包涵体进行变性溶解,经镍柱亲和纯化得到纯度超过95%的变性蛋白.尝试透析复性、稀释复性和柱上复性3种方法分别对p53变性蛋白进行复性.结果表明透析复性的复性率最高,这为原核表达制备p53提供了一种参考.本研究为ELISA法检测血清中p53蛋白及其抗体诊断肿瘤相关试剂盒研发奠定了基础.

Expression,Purification and Renaturation of Human P53

The preparation of p53 protein was foundation of the development of ELISA kit to diagnose tumor by detection of serum p53 protein and its antibody.Through construction of p53 prokaryotic expression vectors pET-32a-p53,induced the expression,denaturation of inclusion bodies,purification,and renaturation,soluble recombinant protein of p53 was produced.Results showed denatured protein of p53 more than 95% purity was got through the denaturation of the inclusion bodies with 8Murea,purified by Ni-affinity chromatography.To renature it,Ni-NTA affinity renaturation,dilution renaturation,and dialysis renaturation were used,in which dialysis renaturation came to the best result.The study provides a reference which p53 was prepared by the expression of prokaryotic organism and lays foundation of the development of ELISA kit to diagnose tumor by detection of serum p53 protein and its antibody.