【摘要】 将纤溶酶原激活剂抑制物 2型 (PAI 2 )突变体PAI 2CD和PAI 2QcDNA与真核表达载体 pcDNA3重组 ,构建真核表达质粒并转染HeLa细胞 ,经G4 18筛选 ,Northern印迹鉴定和ELISA检测 ,筛选出稳定表达PAI 2CD和PAI 2Q的阳性细胞株。ELISA结果表明PAI 2突变蛋白质在转染细胞中的表达量与相应野生型PAI 2在阳性细胞克隆中的表达量基本相近 ,PAI 2及其突变体在理化、生物学性质上也基本一致 ,而结晶紫染色、MTT法证实PAI 2突变体均未能抑制肿瘤坏死因子 α(TNF α)的细胞毒性作用。结果表明PAI 2抑制TNF α诱导的细胞凋亡作用依赖PAI 2螺旋CD间的环状结构所介导的蛋白质与蛋白质间的作用 ;而谷氨酰胺转移酶 (tTG)所介导的PAI 2与其他胞内蛋白质的相互作用可能是凋亡抑制中的关键作用。更多还原

【Abstract】 The cDNA of PAI 2 mutants, PAI 2CD and PAI 2Q were inserted into eukaryotic expression vector, pcDNA3, producing pZLE PAI 2CD and pZLE PAI 2Q respectively. The PAI 2 mutant expression plasmids were transfected into HeLa cells and the transfected cells expressing mutant proteins were selected by G418, Northern blot and ELISA assay. The ELISA assay showed that the expression level of PAI 2 mutant protein was equal to that of wild type PAI 2 in transfected cells. The biochemical characteristics of PAI 2 mutant proteins were very similar to that of the wild type PAI 2 . The results of crystal violet and MTT assays showed that the PAI 2 mutants did not protect cells from the cytotoxic effects of TNF α. The results of this study indicate that the protection of PAI 2 from apoptosis induced by TNF α is dependent upon the protein protein interaction mediated by the CD interhelical domain of PAI 2. The tTG mediated interaction between PAI 2 and cytosol protein may play a leading role in protecting from apoptosis induced by TNF α.更多还原