摘要
心肌肌钙蛋白I(cTnI)是检测心肌损伤的理想标志物 ,具有很高的特异性 ,在病人体内可停留较长时间 .以人心肌cDNA为模板 ,通过PCR方法克隆了人的cTnI和cTnC基因 ,将cTnI和cTnC基因插入到原核表达载体 pBV2 2 0中 ,转化E .coliDH5α菌株并诱导表达 ,经SDS PAGE分析 ,分别发现分子质量为 2 8ku和 18ku的特异性蛋白条带 .cTnI经Westernblotting检测 ,结果与天然蛋白一致 .将纯化得到的cTnI免疫小鼠后 ,用人天然标准抗原检测小鼠的抗血清效价在 1∶16 0 0 0以上 ,并且呈高度特异性 ,与cTnC无交叉反应 .而表达的cT nC可以在一定条件下和cTnI结合 ,证明 2种基因工程蛋白是具有天然构象和结合功能的 .cTnI和cTnC的表达及 2种蛋白结合试验的研究结果为进一步研发免疫诊断试剂奠定了良好的基础 .
The level of cardiac troponin I is a gold indicator for detecting cardial injury because of its specifity. The genes of cardiac troponin I and C subunits were cloned into pBV220 vector and expressed in E. coli DH5 α respectively. Recombinant proteins of cTnI and cTnC were identified by SDS-PAGE. Two protein bands of 28 ku and 18 ku, were identified as correspondent with the predicted M of cTnI and cTnC, respectively. The recombinant proteins were also confirmed by western blotting using standard cTnI as control. The anti-serum obtained by immunizing Balb/c mice with the purified cTnI can recognize standard cTnI extracted from human heart even diluted more than 16 000 times. At the same time, recombination assay of cTnI and cTnC proteins showed that the two proteins could recognize each other and combine together at the presence of Ca^(2+) and Mg^(2+) as cofactors.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
2004年第2期175-180,共6页
Journal of Fudan University：Natural Science
关键词
心肌肌钙蛋白
心肌梗塞
原核表达
cardiac troponin
myocardial infarction
prokaryotic expression
