亲和层析法提取纯化人心肌肌钙蛋白I

Extraction and purification of human cardiac troponin I by affinity chromatography

目的研究出一种提纯人心肌肌钙蛋白I(cTnI)的简便方法。方法将兔骨骼肌肌钙蛋白C(sTnC)与经溴化氰活化的Sepharose 4B凝胶偶联,制成sTnC亲和层析柱。人心肌经匀浆、离心后,上清液上sTnC亲和层析柱,洗脱后可获得cTnI纯品。结果SDS-PAGE得到一条电泳带,其分子量为25 kD。Western blot结果表明,提纯的cTnI可被cTnI单克隆抗体特异性识别。cTnI产率为82.6 mg/100 g人心肌。结论亲和层析法提高了cTnI的产率,为下一步cTnI体外诊断的研究工作打下基础。

Objective To develop a simple method to purify human cardiac troponin Ⅰ（cTnⅠ）. Methods The affinity chromatography gel was prepared by binding rabbit skeletal troponin C （sTnC） on the CNBr-act Sepha- rose 4B. Human cardiac muscle was homogenized and centrifuged, then the supemate flew through the affinity chromatography gel. The purified cardiac troponinⅠ was isolatod after the elution. Results The purifiod cTnⅠ showod single band by SDS-PAGE analysis, and the molecular weight was 25kD. Western blot analysis showed that the purifiod cTnⅠ can be recognized specifiod by cTnⅠ monoclonal antibody. The yield was 82.6 mg/100 g human cardiac tissue. Conclusion The method by affinity chromatography increased the yield of cTnⅠ and provides the possibility for cTnⅠ immunoassay in vitro.