重组人肝刺激因子非融合性蛋白表达载体的构建及其在大肠杆菌中的表达和纯化

目的构建人肝刺激因子(human hepatic stimulator substance,hHSS)体外表达体系。方法hHSS基因片段经PstⅠ和NdeⅠ消化后,应用T-A克隆技术,克隆到质粒pT7-7载体,通过限制性酶切图谱分析及DNA测序反应鉴定hHSS基因。再将pT7-7-hHSS表达载体转化到BL21(DE3)中表达,通过超滤膜及阴离子交换柱纯化,获得目的蛋白。结果表达产物在15 000处有一明显条带,蛋白质肽指纹图谱分析与预期结果相符,经Western blot杂交印证,该15 000蛋白质为hHSS。结论通过构建pT7-7-hHSS原核表达体系,能正确高效地表达hHSS。

Recombination and Expression, Purification of Human Hepatic Stimulatory Substance by Non-fusion Vector in E. coli

Objective To establish the prokaryotic expression system for expression of human hepatic stimulator substance.Methods Fulllength of hHSS cDNA was digested by PstⅠand NdeⅠand the fragment of open reading frame(ORF) was inserted into pT7-7 vector.The construct of prokaryotic expression vector pT7-7-hHSS identified by endonuclease digestion and DNA sequencing.Recombinant hHSS was purified by microcon centrifugal filter.The hHSS was identified by Western blot and Peptide Mass Fingerprint.Results There was one obvious band at the position of relative molecular weight(150000),which was positively reacted with anti-HSS antibody in Western blot.The sequencing of such(150000) protein indicated that the amino acids was equivalent to the deduced data.Conclusion The recombinant fusion express vector pT7-7-hHSS is constructed,and hHSS gene is expressed in E. coli successfully.