人血管内皮抑素的基因克隆和表达载体构建及纯化

Cloning, Construction and Purification of Recombinant Human Endostatin Gene

Objective To clone human endostatin gene, purify its expression protein and detect its biological activity. Methods By using PCR technique, the gene encoding human endostatin was cloned from the cDNA library of the adult human liver and sequenced. Then this gene was inserted into expression vector pET -22b( + ). The construct was expressed in E. coli and the expression product was purified by affinity chromatography through Ni + - IDA Sepharose 6B. The purified protein was tested by en-dothelial cell proliferation inhibitory assay. Results The acquired endostatin gene was 551 bp and its sequence was correct. The construct was expressed in E. coli with a high level as soluble protein, accounting for 10% of the total bacterial proteins. This gene product, characterized by SDS - PAGE and Western blot appeared to be a protein with molecular mass of 21kd. The purity of endostatin -6xHis purified by affinity chromatography reached more than 90% . The protein possesses the inhibitory activity of endothelial cell proliferation. Conclusion The cloning, expression and purification of human endostatin protein lay the foundation for antiangiogenesis therapy in tumor bearing nude mice and tumor patients.