重组人心肌肌钙蛋白I基因工程菌的构建与表达

摘要

Human cardiac troponin I(hcTnI)is an important biochemical marker for myocardial injury and prognosis of myocardial injury. However,it is hard to prepare it in quantity. In order to obtain unlimited hcTnI protein,we constructed genetic bacteria to produce the protein. In this research,the gene of human cardiac troponin I was synthesized by a biochemi-cal company. Then,the commercial genes were inserted into the vector pET-11a in order to construct a high efficiency ex-pression system inE.coli BL21. The recombined plasmid was indentified by the digestion of restriction endonucleases. Fi-nally,the recombined purpose protein was expressed and identified by the Western Blot Assay. The relative molecular weight of hcTnI was about 2.6×104 identified by SDS-PAGE,which was the same as the reported data. The expressed ac-tive rhcTnI protein was obtained and amounted to 30.1% of the total bacterial proteins as detected with the densitometer scan software. The immunological activity of the expressed rhcInI was analyzed by Western Blot Assay after SDS-PAGE,and the result indicated that the recombined protein has good immunologic affinity. The recombinedhuman cardiac troponin I was successfully expressed on a large scale.It can be used in producing monoclonal antibody and also contribute to the research of hcTnI diagnosis standardization.%人心肌肌钙蛋白I(hcTnI)是临床检测心肌损伤及预后提供诊断的生物学指标，由于来源有限，采用基因重组技术，以期获得高表达量的人心肌肌钙蛋白 I．人工合成 hcTnI 基因，将其插入 pET-11a 载体中，通过酶切鉴定正确后转入表达宿主菌BL21(DE3)中，诱导表达目的蛋白．采用蛋白免疫印迹反应(Western Blot，WB)鉴定表达目的蛋白的免疫特异性．经SDS-PAGE证实重组蛋白的相对分子质量约为2.6×104，凝胶密度扫描软件检测到目的蛋白占总蛋白比例为30.1%，WB实验验证诱导后目的蛋白特异性良好．成功构建了重组hcTnI基因在大肠杆菌表达的工程菌株，并获得表达，为制备高特异性的抗体及临床检测应用和测定标准化奠定了基础．