ABSTRACT
ObjectiveTo optimize human Hexastatin gene,to express,purify protein and conduct activity experimental research,and to provide a theoretical basis for further study of Hexastatin.MethodsHuman Hexastatin gene was optimized and synthesized.It was connected to the pET28a expression vector,induced to express by isopropyl β-D-1-thiogalactopyranoside(IPTG),and optimized induction conditions.After the ultrasonication of bacterial cells and inclusion bodies,the recombinant fusion protein was purified with Ni-NTA chromatographic column,analyzed and identified by SDS-PAGE and Western Blot,and conduct activity experimental research in vitro by MTT.ResultsConstructed production was pET28a-Hexastatin expression plasmid.The human Hexastatin protein was expressed in E.coli BL21 the high level and accounted for 45.1% of the total bacterial protein.The purification of recombinant protein purified with Ni-NTA chromatographic column reached 90%,and the concentration was 80 μg/ml.Human Hexastatin protein can restrain the growth of C6,MCF-7 and human vascular endothelial cell (HMEC) cells,and inhibition ratio reach to 72.9%±3.6%,48.8%±2.9%,52.7%±2.5%,respectively through MTT test.ConclusionThe optimized human Hexastatin protein was expressed successfully,which confirmed the inhibition to tumour cells.It is a new way for anti-angiogenesis therapy of tumour.