目的:克隆和构建含有肿瘤坏死因子-α转换酶金属蛋白酶区(m etalloprote inase dom ain of hum an tumor necrosis factor-αconverting enzym e,TACEmp)基因表达载体,并在大肠埃希菌中高效表达。方法:用RT-PCR方法,从THP-1细胞中扩增出...目的:克隆和构建含有肿瘤坏死因子-α转换酶金属蛋白酶区(m etalloprote inase dom ain of hum an tumor necrosis factor-αconverting enzym e,TACEmp)基因表达载体,并在大肠埃希菌中高效表达。方法:用RT-PCR方法,从THP-1细胞中扩增出TACEmp基因片段,插入表达载体pET-D sbAmut,用限制性酶切和DNA测序进行鉴定;转化到大肠埃希菌BL21中进行诱导表达,通过SDS-PAGE电泳分析表达产物。结果:克隆TACEmp基因并构建重组表达载体pET-D sbAmut-TACEmp转化入BL21,IPTG诱导后获得高效表达的TACEmp融合表达蛋白。结论:利用分子伴侣融合蛋白技术使TACEmp在原核表达系统中获得了高效可溶性表达。展开更多
在许多生理过程中,细胞内的氧化还原状态(Redox)与非折叠蛋白反应(Unfolded protein response UPR)对于细胞的生存和死亡有着重要的意义。本文探讨了UPR的氧化还原机制,其中包括蛋白质二硫键异构酶与ER应激的直接相互作用,Redox与钙离...在许多生理过程中,细胞内的氧化还原状态(Redox)与非折叠蛋白反应(Unfolded protein response UPR)对于细胞的生存和死亡有着重要的意义。本文探讨了UPR的氧化还原机制,其中包括蛋白质二硫键异构酶与ER应激的直接相互作用,Redox与钙离子外流,细胞的氧化还原能力与UPR信号通路。展开更多
Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins （n 〉 2） in rabbits with spinal cord ...Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins （n 〉 2） in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initially improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradually decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6-12 hours, showing a characterization of induction-inhibition-induc- tion. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons.展开更多
文摘目的:克隆和构建含有肿瘤坏死因子-α转换酶金属蛋白酶区(m etalloprote inase dom ain of hum an tumor necrosis factor-αconverting enzym e,TACEmp)基因表达载体,并在大肠埃希菌中高效表达。方法:用RT-PCR方法,从THP-1细胞中扩增出TACEmp基因片段,插入表达载体pET-D sbAmut,用限制性酶切和DNA测序进行鉴定;转化到大肠埃希菌BL21中进行诱导表达,通过SDS-PAGE电泳分析表达产物。结果:克隆TACEmp基因并构建重组表达载体pET-D sbAmut-TACEmp转化入BL21,IPTG诱导后获得高效表达的TACEmp融合表达蛋白。结论:利用分子伴侣融合蛋白技术使TACEmp在原核表达系统中获得了高效可溶性表达。
文摘在许多生理过程中,细胞内的氧化还原状态(Redox)与非折叠蛋白反应(Unfolded protein response UPR)对于细胞的生存和死亡有着重要的意义。本文探讨了UPR的氧化还原机制,其中包括蛋白质二硫键异构酶与ER应激的直接相互作用,Redox与钙离子外流,细胞的氧化还原能力与UPR信号通路。
基金Supportedby Major State Basic Research Development Program of China(973 Program,No.2006CB101700)National High Technology Research and Development Programof China(863 Program,No.20060110Z1141)National Natural Science Foundation of China(No.30571133)~~
基金supported by the National Natural Science Foundation of China, No. 30872609
文摘Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins （n 〉 2） in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initially improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradually decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6-12 hours, showing a characterization of induction-inhibition-induc- tion. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons.
