人心肌肌钙蛋白Ⅰ在大肠杆菌中的高效表达及纯化

目的:获得高纯度的人心肌肌钙重组蛋白,为临床上检测心肌损伤及预后提供新的诊断方法.方法:利用RT-PCR方法从人心肌细胞的cDNA文库中扩增编码人心肌肌钙蛋白Ⅰ的cDNA,并克隆到大肠杆菌表达载体中,表达含凝血酶识别序列的MS2-cTnI 融合蛋白,纯化后用Western-blot进行鉴定.结果:从人心肌cDNA文库中扩增出编码人cTnI的cDNA,克隆并在大肠杆菌中表达,表达量达30%以上.经离子交换柱纯化,最终产物纯度在95%以上.可与其特异性单克隆抗体反应.结论:cTnI能够在大肠杆菌中高效表达和纯化,为今后检测心血管疾病提供了一种高效快捷的方法.

The expression and purification of human cTnI overexpressed in E.coli

Objective: To obtain and purify rhcTnI. Methods: cDNA encoding cTnI was amplified from heart muscle cDNA library by PCR and then subcloned into prokarytic vector with a thrombin linker. MS2 cTnI fusion protein was expressed in E.coli . Results: Up to 30% of total bacterial proteins were rhcTnI as shown in SDS PAGE gel. The rhc TnI was purified to 95% homogeneity. Conclusion: rhc TnI can be expressed in E.coli in high quantity and purified to hear homogeneity.