cTnI-linker-TnC融合蛋白基因的构建、表达及鉴定

目的重组及表达人cTnI-linker-TnC融合蛋白。方法应用PCR技术从人心脏cDNA文库分别扩增出cTnI和TnC基因,通过引物设计在两者之间加上19个中性氨基酸残基TS-(G4S)3-AC的linker编码序列,克隆PCR产物,并构建pET28a-cTnI-linker-TnC表达质粒,转化入大肠杆菌表达菌株BL21(DE3)中,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-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21 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可以获得具有高纯度和高免疫反应活性的cTnI-linker-TnC融合蛋白。

Construction,Expression of cTnI-linker-TnC Fusion Protein and its Identification

Objective To construct cardiac TnI-linker-TnCgene,purify its expression and study its immunactivity.Methods The gene encoding cardiac troponin I and troponin C were cloned from the human heart quick-clone cDNA by using PCR technique.The cTnI was linked with TnCby a short DNA sequence coding for 19 neutral amino acid residues.An expressionconstruct for cTnI-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 bepurified by affinity chromatography on a NTA-Sepharose column,then judged its immunoreactivity.Results Soluable expression of cTnI-linker-TnC in prokaryotic system was successfully obtained.Fusion protein had an N-terminal His-tag sequence which could be purified by affinity chromatographyon 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-linker-TnC with high purity and immunoreactivity could be successfully obtained by expression in prokarayotic.