A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eu...A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.展开更多
研究利用intein的蛋白质反式剪接功能在大肠杆菌中对凝血VIII因子(FVIII)重链和轻链的连接作用,将B结构域大部分缺失型FVIII(BDD-FVIII)于满足剪接所需的保守性氨基酸Ser1657前断裂为重链和轻链,分别与split mini Ssp DnaB intein的106...研究利用intein的蛋白质反式剪接功能在大肠杆菌中对凝血VIII因子(FVIII)重链和轻链的连接作用,将B结构域大部分缺失型FVIII(BDD-FVIII)于满足剪接所需的保守性氨基酸Ser1657前断裂为重链和轻链,分别与split mini Ssp DnaB intein的106个氨基酸的N端(Int-N)和48个氨基酸的C端(Int-C)融合,构建到原核表达载体pBV220。诱导表达后SDS-PAGE分析可见预期大小的BDD-FVIII蛋白条带,Western blotting用FVIII特异性抗体证明其为剪接所产生的BDD-FVIII蛋白,表明intein可有效连接BDD-FVIII的重链和轻链。为进一步甲型血友病基因治疗研究应用intein以双腺相关病毒载体(AAV)携带FVIII基因,克服单个AAV载体的容量限制提供了依据。展开更多
Background and objective Metastasis is one of the most important causes of mortality in tumor. The pathological process of metastasis includes several sequential steps as
基金supported by the Natural Science Foundation of Shandong Province, China (Grant No. Y2005D14)the Science and Technology Program of Yantai (Grant No. 2008152)+1 种基金the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (Grant No. 20071108)the Discipline Construction Funds of Ludong University
文摘A dual-vector system was explored for the delivery of the coagulation factor VIII gene,using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally.A pair of eukaryotic expression vectors,expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII),was constructed.With transient co-transfection of the two vectors into 293 and COS-7 cells,the culture supernatants contained (137±23) and (109±22) ng mL–1 spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL–1 for 293 and COS-7 cells,respectively.The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes.The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting.The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.
基金supported partially by Natural Science Foundation of Shandong Province (Y2005D14)Science and Technology Program of Yantai City (2008152)the Scientic Research Foundation from Education Ministry for the Returned Overseas Chinese Scholars and the Scientic Research Foundation of Ludong University
文摘Background and objective Metastasis is one of the most important causes of mortality in tumor. The pathological process of metastasis includes several sequential steps as
基金Supported by National Science and Engineering Research Council grant of CanadaNational High Technology Research and Development Program of China(863 Program,No.2006AA03Z451)+2 种基金National Natural Science Foundation of China(No.31070698)Shanghai Key Projects of Basic Research(No.10JC1400300)the Ph.D.Programs Foundation of Ministry of Education of China(No.20120075110007)~~
