增强的蛋白质反式剪接作用提高肝脏靶向双载体凝血第八因子转基因小鼠的血浆凝血活性
摘要
基于蛋白质反式剪接的双载体转凝血第八因子(FⅧ)基因受到剪接效率低的不利影响,本研究旨在增强蛋白质剪接元件蛋白内含子(intein)之间的相互作用,通过改善蛋白质反式剪接效率提高小鼠体内双载体转FⅧ基因后血浆剪接FⅧ蛋白的分泌量和凝血活性.近期培养细胞水平证明,FⅧ重链和轻链分别进行Cys点突变(Met226Cys和Asp1828Cys)后,可形成链间二硫键,并提高蛋白质剪接效率产生更多的FⅧ蛋白.在此基础上,将蛋白内含子融合到含Cys点突变的人FⅧ重链和轻链基因,构建一对表达载体,门静脉注射C57BL/6小鼠进行肝脏靶向转基因.48h后分别检测小鼠血浆中分泌的人FⅧ蛋白量和由其所产生的凝血活性,结果显示FⅧ重链抗原分泌量为(442±151)ng/mL,凝血活性为(1.46±0.37)IU/mL,接近于单载体转FⅧ基因产生的血浆凝血活性((1.79±0.59)IU/mL),明显高于双载体转FⅧ基因对照小鼠血浆的重链分泌量((305±103)ng/mL)和血浆凝血活性((0.85±0.23)IU/mL).结果表明,链间二硫键交联可通过改善蛋白质反式剪接效率提高双载体转FⅧ基因的功效.
引文
1朱甫祥,刘泽隆,迟晓艳,等.基于蛋白质反式剪接的双载体凝血Ⅷ因子基因转移.中国科学C辑,2009,39:746-754
2Wu H,Xu M Q,Liu X Q.Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein.Biochim Biophys Acta,1998,1387:422-432
3Scallan C D,Liu T,Parker A E,et al.Phenotypic correction of a mouse model of hemophilia A using AAV2vectors encoding the heavy and light chains of FVIII.Blood,2003,102:3919-3926
4Zhang K,Shen X,Wu J,et al.Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response.Cell,2006,124:587-599
5朱甫祥,刘泽隆,缪静,等.Met662和Asp1828的Cys突变增强蛋白内含子对共表达的BDD-FVIII重链和轻链的剪接.中国生物化学与分子生物学报,2012,28:234-239
6Burton M,Nakai H,Colosi P,et al.Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein.Proc Natl Acad Sci USA,1999,96:12725-12730
7Chen L X,Zhu F X,Li J,et al.The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene.Mol Ther,2007,15:1856-1862
8Swaroop M,Moussalli M,Pipe S W,et al.Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII.J Biol Chem,1997,272:24121-24124
9Arruda V R,Stedman H H,Haurigot V,et al.Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.Blood,2010,115:4678-4688
10Niemeyer G P,Herzog R W,Mount J,et al.Long-term correction of inhibitor-prone hemophilia B dogs treated with liverdirected AAV2-mediated factor IX gene therapy.Blood,2009,113:797-806
11Sarkar R,Mucci M,Addya S,et al.Long-term efficacy of adeno-associated virus serotypes8and9in hemophilia a dogs and mice.Hum Gene Ther,2006,17:427-439
12Kaufman R J,Wasley L C,Dorner A J.Synthesis,processing,and secretion of recombinant human factor VIII expressed in mammalian cell.J Biol Chem,1988,263:6352-6362
13Sabatino D S,Lange A M,Altynova E S,et al.Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors.Mol Ther,2011,19:442-449
14Mah C,Sarkar R,Zolotukhin I,et al.Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice.Hum Gene Ther,2003,14:143-152
15Pipe S W.Coagulation factors with improved properties for hemophilia gene therapy.Semin Thromb Hemost,2004,30:227-237
16Miao H Z,Sirachainan N,Palmer L,et al.Bioengineering of coagulation factor VIII for improved secretion.Blood,2004,103:3412-3419
17Gale A J,Radtke K P,Cunningham M A,et al.Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.J Thromb Haemost,2006,4:1315-1322
18朱甫祥,刘泽隆,缪静,等.链间二硫键改善FⅧ基因断裂转移小鼠血浆重链分泌和凝血活性.中国科学:生命科学,2012,42:538-543
19朱甫祥,杨树德,刘泽隆,等.分泌改善的重链促进基于蛋白内含子的双载体共转全长FVIII基因.科学通报,2010,55:3199-3204
20Kaufman R J.Genetic engineering of factor VIII.Nature,1989,342:207-208
21Vila-Perello M,Muir T W.Biological applications of protein splicing.Cell,2010,143:191-200
2Wu H,Xu M Q,Liu X Q.Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein.Biochim Biophys Acta,1998,1387:422-432
3Scallan C D,Liu T,Parker A E,et al.Phenotypic correction of a mouse model of hemophilia A using AAV2vectors encoding the heavy and light chains of FVIII.Blood,2003,102:3919-3926
4Zhang K,Shen X,Wu J,et al.Endoplasmic reticulum stress activates cleavage of CREBH to induce a systemic inflammatory response.Cell,2006,124:587-599
5朱甫祥,刘泽隆,缪静,等.Met662和Asp1828的Cys突变增强蛋白内含子对共表达的BDD-FVIII重链和轻链的剪接.中国生物化学与分子生物学报,2012,28:234-239
6Burton M,Nakai H,Colosi P,et al.Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein.Proc Natl Acad Sci USA,1999,96:12725-12730
7Chen L X,Zhu F X,Li J,et al.The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene.Mol Ther,2007,15:1856-1862
8Swaroop M,Moussalli M,Pipe S W,et al.Mutagenesis of a potential immunoglobulin-binding protein-binding site enhances secretion of coagulation factor VIII.J Biol Chem,1997,272:24121-24124
9Arruda V R,Stedman H H,Haurigot V,et al.Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.Blood,2010,115:4678-4688
10Niemeyer G P,Herzog R W,Mount J,et al.Long-term correction of inhibitor-prone hemophilia B dogs treated with liverdirected AAV2-mediated factor IX gene therapy.Blood,2009,113:797-806
11Sarkar R,Mucci M,Addya S,et al.Long-term efficacy of adeno-associated virus serotypes8and9in hemophilia a dogs and mice.Hum Gene Ther,2006,17:427-439
12Kaufman R J,Wasley L C,Dorner A J.Synthesis,processing,and secretion of recombinant human factor VIII expressed in mammalian cell.J Biol Chem,1988,263:6352-6362
13Sabatino D S,Lange A M,Altynova E S,et al.Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors.Mol Ther,2011,19:442-449
14Mah C,Sarkar R,Zolotukhin I,et al.Dual vectors expressing murine factor VIII result in sustained correction of hemophilia A mice.Hum Gene Ther,2003,14:143-152
15Pipe S W.Coagulation factors with improved properties for hemophilia gene therapy.Semin Thromb Hemost,2004,30:227-237
16Miao H Z,Sirachainan N,Palmer L,et al.Bioengineering of coagulation factor VIII for improved secretion.Blood,2004,103:3412-3419
17Gale A J,Radtke K P,Cunningham M A,et al.Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.J Thromb Haemost,2006,4:1315-1322
18朱甫祥,刘泽隆,缪静,等.链间二硫键改善FⅧ基因断裂转移小鼠血浆重链分泌和凝血活性.中国科学:生命科学,2012,42:538-543
19朱甫祥,杨树德,刘泽隆,等.分泌改善的重链促进基于蛋白内含子的双载体共转全长FVIII基因.科学通报,2010,55:3199-3204
20Kaufman R J.Genetic engineering of factor VIII.Nature,1989,342:207-208
21Vila-Perello M,Muir T W.Biological applications of protein splicing.Cell,2010,143:191-200