Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) vectors.Inhibited sec...Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) vectors.Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain.Previously,we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene,suggesting that the light chain,covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII.However,its level of secretion was affected by inefficient secretion the heavy chain.Here,we studied the effect of a mutant heavy chain with L303E/F309S substitutions,which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene.Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells.A spliced FVIII protein was seen in co-transfected cells by Western blot analysis.The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene,which was greater than the secretion of wild-type heavy chain.The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL,with an activity of (0.61±0.08) IU/mL,which was greater than that of wild-type FVIII co-transfected cells.Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections.This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector.These results encourage the transfer of this technology 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
基金supported by the Natural Science Foundation of Shandong Province,China(ZR2010CM061)the Science and Technology Program of Yantai(2008152)the Scientific Research Foundation of Ludong University(LZ20083305)
文摘Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene,which is difficult to package in the promising adeno-associated virus (AAV) vectors.Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain.Previously,we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene,suggesting that the light chain,covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII.However,its level of secretion was affected by inefficient secretion the heavy chain.Here,we studied the effect of a mutant heavy chain with L303E/F309S substitutions,which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene.Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells.A spliced FVIII protein was seen in co-transfected cells by Western blot analysis.The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene,which was greater than the secretion of wild-type heavy chain.The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL,with an activity of (0.61±0.08) IU/mL,which was greater than that of wild-type FVIII co-transfected cells.Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections.This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector.These results encourage the transfer of this technology
