【摘要】 目的构建及表达人cTnI(28-110 aa)-linker-TnC融合蛋白。方法应用PCR技术从人心脏cDNA文库中扩增出cTnI(28-110 aa)和TnC基因,通过引物设计在两者之间加上linker序列即编码19个中性氨基酸残基TS-(G4S)3-AC的序列,克隆PCR产物,并构建成pET28 a-cTnI(28-110 aa)-lin-ker-TnC表达质粒,转化入大肠杆菌表达菌株BL21(DE3),用异丙基硫代-β-D-半乳糖苷(IPTG)诱导目的蛋白的表达,NTA树脂亲和层析纯化后检测其纯度和免疫反应性。结果成功构建了cTnI(28-110 aa)-linker-TnC融合蛋白的基因,并在大肠杆菌中实现可溶性高表达,在摇瓶中的表达量为20 mg/L,经一步NTA树脂亲和层析纯化,获得条带单一的目的蛋白,采用进口全自动免疫检测系统鉴定证实,目的蛋白有较高的免疫反应性。结论采用原核表达方法获得了具有高纯度和高免疫反应活性的cTnI(28-110 aa)-linker-TnC融合蛋白。更多还原

【Abstract】 Objective To construct cardiac troponin I(28-110aa)-linker-TnC gene,purify its expression and study its immunoactivity.Methods The gene encoding cardiac troponin I(28-110aa) and troponin C were cloned from the Human Heart Quick-Clone cDNA by using PCR technique.The cTnI(28-110aa) was linked with TnC by a short DNA sequence coding for 19 neutral amino acid residues.An expression constructed for cTnI(28110aa)-linker-TnC was engineered by inserting the corresponding DNA into a pET28a plasmid.Then recombinant plasmid was transformed into E.coli BL21(DE3 ) cells,and protein expression was induced by IPTG.Fusion protein was purified by affinity chromatography on a NTA-Sepharose column,then judging its immunoreactivity.Results Soluable expression of cTnI(28-110aa)-linker-TnC in prokaryotic system was successfully obtained.Fusion protein had anN-terminal His-tag sequence which could be purified by affinity chromatography on a NTA-Sepharose column.After one step affinity chromatography the fusion protein shows homogeneity as judged by SDS-PAGE and high immunoreactivity by random access chemiluminescent system.Conclusion The fusion protein cTnI(28-110aa)-linkerTnC with high purity and immunoreactivity was successfully obtained by expression in prokarayotic.更多还原