人HCCR蛋白表达载体的构建、表达及其蛋白纯化

目的:构建人HCCR蛋白表达载体,并探讨其体外表达及表达产物的纯化。方法:培养人肝癌细胞,提取总RNA,反转录合成cDNA第一链,以此cDNA为模板经PCR合成插入片段,连接至载体pMBP-C,转化到大肠杆菌Top10F′中,进行诱导表达。表达产物通过Ni-NTA螯合层析进行纯化。经SDS-聚丙烯酰胺凝胶电泳,Westernblot和电喷雾电离串联飞行时间质谱(ESI-TOFMS)鉴定表达产物。结果:构建的表达载体经限制性内切酶酶切分析和DNA测序,证明所构建的质粒含有HCCR基因。SDS-聚丙烯酰胺凝胶电泳发现该重组质粒经IPTG诱导后表达一种新的蛋白,这种蛋白不存在于诱导后的空载体表达产物中,表达产物经Westernblot和蛋白质谱鉴定含有HCCR蛋白的部分肽段。结论:成功构建了HCCR蛋白表达载体并进行体外表达及纯化。

Construction, expression and purification of plasmid containing HCCR protein

Objective:To construct a recombinant vector containing human HCCR.Methods:The COOH terminus of its cDNA encoding a polypepide from 167 to 360 amino acid residues was cloned into the pMBP-C vector.The recombinant plasmid was transformed into E.coli Top10F'.The corrected clone identified by endonuclease digestion and nucleotide sequencing was induced for expression.The expression product was purified by nick-nitrilotriacetic acid(Ni-NTA)metal-affinity chromatography and analyzed by SDS-PAGE,western blot and electrospmy ionization time-of-flight mass spectrometry.Results:Nucleotide sequencing showed there was no sense mutation.A 67 kD protein was expressed after induction and purification.Conclusion:A recombinant plasmid containing HCCR167-360 fragment is successfully constructed and expressed.