基因工程人抗体Fab片段的纯化及鉴定

Purification and identification of human antibody Fab expressed in E.coli.

目的 通过亲和层析的方法纯化基因工程人抗体Fab(Fragment,antigenbinding)片段。方法 对可以表达人抗体Fab的菌种进行诱导 ,冻融裂菌离心收集上清 ,进行亲和层析并对纯化前后的样品进行电泳分析及Westernblot检测。结果 SDS -PAGE电泳显示纯化品在还原、非还原条件下均为单一条带。Westernblot分析层析前抗体Fab片段还原的分子量在 2 .4× 10 4 左右 ,非还原的分子量约为 4.8× 10 4 ,而经过亲和层析后 ,在凝胶电泳上纯化品在还原、非还原条件下在相应位置上均显示单一蛋白带。结论 我们可以经过亲和层析 。

Objective Antigen binding fragments (Fab) were purified by affinity chromatography.Methods The bacterial strains for expressing Fab were induced,and the supernates were collected by centrifuging the lysates of strains.Then,the Fab contained in supernates was purified by affinity chromatogriaphy.Finally,SDS-PAGE gel electrophoresis and Western blot of Fab were done with or without β-mercaptoethanol in the sample.Results Before chromatography the reduced Fab was 2.4×10 4 Dalton MW,while the nonreductant was 4.8×10 4 Dalton MW.A single protein band still existed in the right position at SDS-PAGE gel and Western blot,after the Fab was purified.Conclusions The affinity chromatography can be used in the purification of Fab.