内含肽介导三段vWF基因真核细胞转移的翻译后连接及其功能性多聚体形成  

Post-translational Ligation and Functional Multimerization of von Willebrand Factor After Intein-mediated Tri-fragmental Gene Delivery in Eukaryotic Cell

作　　者：朱甫祥[1] 杨树德[1] 刘泽隆[1] 缪静[1] 屈慧鸽[1] 迟晓艳[1] 

机构地区：[1]鲁东大学生命科学学院,烟台264025

出　　处：《生物化学与生物物理进展》2011年第1期67-74,共8页Progress In Biochemistry and Biophysics

基　　金：山东省自然科学基金(ZR2010CM061);烟台市科技发展计划(2008152);教育部留学回国人员科研启动基金(20071108);鲁东大学学科建设经费资助项目~~

摘　　要：vWF(von Willebrand factor)是一种超大分子质量的血浆多聚体糖蛋白,在血栓形成和生理凝血过程中发挥重要作用,其质和/或量的缺陷导致血管性血友病(VWD),由于VWD为单基因病,且vWF为分泌性蛋白,基于基因转移的基因治疗无需特异的靶器官,因此VWD特别适合于基因治疗,但vWF基因过大(8.4 kb),难以为多数病毒载体特别是优点较多的腺相关病毒(AAV)载体承载.运用内含肽(intein)的蛋白质反式剪接功能,研究了三重载体真核细胞共转断裂3段的vWF基因,以期通过转基因翻译后的蛋白质剪接作用形成完整的功能性vWF蛋白.将vWF的cDNA于满足剪接所需的保守性氨基酸Cys1099、Ser2004的密码子前断裂为3段,分别与2种不同的内含肽即Ssp DnaE内含肽和Ssp DnaB内含肽编码序列融合,构建到真核表达载体pcDNA3.1(+),得到3个分别融合内含肽的vWF片段基因真核表达载体,共转染培养的293细胞,通过瞬时表达,电泳观察培养上清中的vWF多聚体形态,分析vWF蛋白量和凝血Ⅷ因子(FⅧ)结合力;通过共转FⅧ基因,分析了培养上清中的FⅧ蛋白量及生物活性.结果显示,通过内含肽的蛋白质反式剪接作用,共转内含肽融合的三片段vWF基因细胞上清,表现与正常人血浆和转vWF基因阳性对照细胞相似的vWF多聚体模式和FⅧ结合力,而且可明显提高转FⅧ基因后表达的FⅧ蛋白的分泌量和活性,提示剪接vWF蛋白的FⅧ载体功能的恢复.结果表明,内含肽可作为一种有效的技术手段进行三重载体共转断裂的vWF基因,为进一步基于内含肽的三重AAV转断裂vWF基因应用于VWD基因治疗研究、克服AAV的容量限制提供了依据.von Willebrand factor（vWF） is a huge multimeric plasma glycoprotein with important functions involved in thrombosis and hemostasis.Its qualitative and/or quantitative abnormalities result in a bleeding disorder termed von Willebrand disease（VWD）.Gene therapy is favorable for treatment of VWD because this disease is monogenic and the vWF is a secretory protein making non-specific targeting organ required for gene delivery.But the vWF gene is hardly packaged in most existing viral vectors especially the desired adeno-associated virus（AAV） vectors for its oversized cDNA in size（8.4 kb）.The intein-mediated protein trans-splicing was explored to co-transfer split three fragmented vWF gene into eukaryotic cells by a ternary-vector system and the functional vWF protein was expected to be formed posttranslationally by protein trans-splicing.The vWF cDNA was broken into three fragments before codons of Cys1099 and Ser2004 which required for protein splicing and then fused with Ssp DnaE and Ssp DnaB inteins respectively.A group of three eukaryotic expression vectors were produced by inserting these three fusion genes into pcDNA3.1（＋） respectively.By transient co-transfection of 293 cells with these three vectors the conditioned culture supernatant was observed for vWF multimer pattern by electrophoresis,analyzed for vWF antigen and binding capacity of coagulation factor Ⅷ（FⅧ） quantitatively.With FⅧ gene co-transfection,the antigen and activity of FⅧ in the culture supernatant were measured.The data showed that with intein-mediated protein trans-splicing after translation the culture supernatant from cells co-transfected with intein-fused three fragmented vWF genes displayed a vWF multimer pattern and FⅧ binding capacity similar to normal human plasma and cells of vWF gene transfected positive control,and the FⅧ secretion and activity were increased dramatically in FⅧ gene co-transfected cells indicating the functional recovery of spliced vWF as a FⅧ carrier.It suggests that inteins

关 键 词：内含肽 蛋白质反式剪接 von WILLEBRAND因子 转基因 多聚体化 FVIII载体 

分 类 号：Q7[生物学—分子生物学]