Molecular Cloning,Expression,Purification,and Characterization of Human Hepatic Stimulatory Substance by Non-fusion Vector of E.coli

Objective With the development of gene techniques,we constructed pT7-7-hHSS(human stimulator substance) prokaryotic expression system.Methods After digestion of pT7-7 and the cDNA of hHSS by PstⅠand NdeⅠ,hHSS gene was ligated into the corresponding sites of the pT7-7,and then transferred into competent cell DH5α.The positive clone DH5α/pT7-7-hHSS was identified by restriction mapping and DNA sequencing.The pT7-7-hHSS was transferred into BL21 host cells,then expressing the target protein induced with IPTG.Recombinant hHSS was purified by Microcon Centrifugal Filter Devices.The hHSS was identified by Western blot and Peptide Mass Fingerprint.The biologic activity of rhHSS was researched by MTT.Results The exogenous purified rhHSS could proliferate the growth of BEL-7402 cells that were a kind of hepatoma cell in a dose-dependent manner.Conclusion In pT7-7-hHSS vector system,hHSS was expressed correctly and quantitatively with activity.