You Position: Home > Paper
Protein trans-spliced chimeric human/porcine BDD-FⅧ with augmented secretion
( views:15, downloads:0 )
- Author:
- No author available
- Journal Title:
- ACTA PHARMACEUTICA SINICA
- Issue:
- 10
- DOI:
- No doi available
- Key Word:
- 嵌合体 BDD-FⅧ;分泌;内含肽;蛋白质反式剪接
Abstract： 本研究采用具有促分泌作用的猪FⅧ分子的A1和A3区,替换人FⅧ分子的相应结构,构建人/猪嵌合体BDD-FⅧ(BDD-hpFⅧ),利用Ssp DnaB intein的蛋白质反式剪接功能,双载体培养的COS-7细胞瞬时共转嵌合体BDD-hpFⅧ基因.免疫印迹法观察了转基因细胞内的蛋白表达和BDD-hpFⅧ的剪接,采用ELISA和Coatest法分别观察了分泌至培养上清液中剪接的嵌合体BDD-hpFⅧ蛋白和生物活性.结果显示,基因共转染细胞内可见明显的剪接BDD-hpFⅧ蛋白,分泌至上清液的剪接BDD-hpFⅧ蛋白量为(340±64)ng·mL-1,活性为(2.52±0.32)u·mL-1,明显高于双载体共转人BDD-FⅧ基因细胞[质量浓度为(93±22)ng·mL-1,活性为(0.72±0.13)u.mL-1],提示intein介导的双载体共转BDD-hpFⅧ基因可明显促进剪接蛋白的分泌.另眄外,分别转intein融合BDD-hpFⅧ重链和轻链基因的细胞混合培养后上清液中仍可检测到剪接的BDD-hpFⅧ蛋白及其活性,分别为(44±9)ng·mL-1和(0.32±0.07)u·mL-1,提示intein可进行不依赖细胞机制的BDD-hpFⅧ剪接.本研究为旨在提高分泌的动物体内应用intein的双载体转BDD-hpFⅧ基因奠定了基础.
- [1]D D, Pittman,K N, Tomkinson,R J, Kaufman.Post-translational requirements for functional factor V and factor VIII secretion in mammalian cells.[J].1994,269.
- [2]Zhu F,Wang X,Arruda VR,etc.The Enhancing Effects of the Light Chain on Heavy Chain Secretion in Split Delivery of Factor VIII Gene.[J].2007,15(10).
- [3]Wang X,Ding Q,Wang J,etc.Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette.[J].2008,19(6).
- [4]F B, Perler,E O, Davis,G E, Dean,etc.Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature.[J].1994,22.
- [5]Ragni MV.Hemophilia gene transfer: comparison with conventional protein replacement therapy.[J].2004,30(2).
- [6]Chong S,Sharma SS,Harcum SW.Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli[J].2006,125(1).
- [7]Dooriss K,Denning G,Doering CB.Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.[J].2009,17(7).
- [8]Healey JF,Doering CB,Lollar P,etc.Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion.[J].2004,279(8).
- [9]Murphy SL,High KA.Gene therapy for haemophilia.[J].2008,140(5).
- [10]Paulmurugan R,Umezawa Y,Gambhir SS.Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies.[J].2002,99(24).
- [11]Mimuro J,Madoiwa S,Kashiwakura Y,etc.Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene.[J].2006,118(5).
- [12]D V, Gnatenko,E L, Saenko,J, Jesty,etc.Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to haemophilia A gene therapy.[J].1999,104(1).
- [13]Spencer HT,Dukart HZ,Gangadharan B,etc.Hematopoietic Stem Cells Encoding Porcine Factor VIII Induce Pro-coagulant Activity in Hemophilia A Mice with Pre-existing Factor VIII Immunity.[J].2007,15(6).
- [14]Cunningham J,Burton M,Nakai H,etc.Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein.[J].1999,96(22).
- [15]朱甫祥,郭猛,刘泽隆,等.双Intein介导3片段vWF的反式剪接[J].2010,(2).