【摘要】 为优化已构建表达人心肌肌钙蛋白I(cTnI)基因工程菌的表达条件,获得纯度较高的cTnI作为抗原用于研制临床上检测心肌梗塞的试剂盒;通过PBD(Plackett Burman Design)设计,考察各因素对目的蛋白cTnI表达的影响,证明诱导时间、pH、KCl浓度是影响最大的三个因素,通过响应曲面法(RSM)优化对cTnI表达影响最大的三个因素,构建回归方程预测cTnI的表达所占总蛋白的百分比,在最优条件下目的蛋白表达所占菌体总蛋白含量的26%。经过硫酸氨沉淀和DEAE-Cellulose离子交换层析,将重组的cTnI蛋白进行纯化,经SDS-PAGE检测后,目的蛋白只有一条带;经Western-blottting和临床检测cTnI试剂盒检测后,证明纯化的cTnI有良好的特性,可以作为免疫动物的抗原,制备特异性强,亲和力好的单克隆抗体,也可以作为临床上标定不同cTnI检测方法的校准物。更多还原

【Abstract】 To optimize the growth condition for the established gene engineer bacteria express cardiac troponin-I(cTnI) and to obtain purified cTnI as an antigen to produce clinical assay kits used in acute myocardial injury(AMI) diagnosis.Plackett Burman Design(PBD) was applied to select the factors which effect the expression of cTnI in Escherichial coli(E.coli) mostly.Induction time,pH and KCl were proved influenced expression of cTnI notably.Afterward,Response Surface Methodology(RSM) as second step to optimize the selected three factors,an equation was deduced to predict the percent of cTnI.In the most optimized condition,the percent of cTnI can reach to 26% of total cell protein.The procedures of purification included ammonium sulfate deposition and DEAE Cellulose ion exchange chromatography.SDS-PAGE shows that purified cTnI contain one band.cTnI could be used to immune animals as an antigen to produce monoclonal antibodies with high affinity and specificity.It maybe as calibrators to harmony the difference assays of cTnI measurement in clinical.更多还原