双Intein介导3片段vWF的反式剪接  

Dual Intein-mediated Trans-splicing of Tri-fragmented von Willebrand Factor

作　　者：朱甫祥[1] 郭猛[1] 刘泽隆[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：《中国生物化学与分子生物学报》2010年第2期157-163,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：山东省自然科学基金(No.Y2005D14);教育部留学回国人员科研启动基金(No.2007-1108);烟台市科技计划项目(No.2008152);鲁东大学学科建设经费资助项目~~

摘　　要：von Willebrand因子(vWF)基因突变导致血管性血友病(VWD),由于其基因过大在基因治疗研究中难以为多数病毒载体携带.利用双内含肽(intein)的蛋白质反式剪接功能研究断裂成3段的vWF基因分别表达后在蛋白水平的连接,旨在为vWF基因的3载体联合转移应用于VWD基因治疗研究提供依据.将vWF 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(N、M和C),分别与splitSspDnaE intein的N端(En)、C端(Ec)和splitSspDnaB intein的N端(Bn)、C端(Bc)编码序列融合,构建到原核表达载体pET-28a(+)中的His-Tag的下游,得到3种表达载体pET-NEn、pET-EcMBn和pET-BcC.分别转化感受态大肠杆菌BL21(DE3)细胞,经IPTG诱导表达后,以SDS-PAGE分析融合蛋白的表达,并进一步用His-Tag的特异性抗体进行分析;亲和层析纯化分别表达的带His-Tag标签的3段蛋白,复性后体外混合进行剪接实验以观察3片段vWF的连接.结果显示,3段预期大小的融合intein的vWF蛋白均有表达,用His-Tag抗体进行的Western印迹得到进一步证实;3段纯化的蛋白混合后可见明显的剪接条带形成,与vWF的预期分子量大小一致,表明双intein通过蛋白质反式剪接可有效连接3个片段的vWF,为进一步应用蛋白质剪接技术的3重载体真核细胞转vWF基因奠定了基础.The mutations of von Willebrand factor（vWF） gene cause von Willebrand disease（VWD）.It＇s difficult to deliver the oversized gene by most of developed viral vectors in gene therapy for VDW.By exploring a dual intein-mediated protein trans-splicing,we investigated the ligation of tri-fragmented vWF after separate expression for further study on delivering this gene by combined triple vectors in VWD gene therapy.The vWF cDNA was broken into three fragments（N-,M-and C-part） before Cys 1099 and Ser 2004 codons which meet the splicing required residues and then fused to N（En） and C terminus（Ec） of split Ssp DnaE and N（Bn） and C terminus（Bc） of split Ssp DnaB inteins,respectively.Three prokaryotic expression vectors pET-NEn,pET-EcMBn and pET-BcC were constructed by inserting these three fusing genes into downstream of His-Tag of pET-28a（＋） vector followed by transformation to competent E.coli BL21（DE3） cells,respectively.By IPTG induction,these three separately expressed recombinant proteins were analyzed by SDS-PAGE followed by further assay with His-Tag antibody-specific Western blotting.After purification and renaturation of these three fusion proteins by affinity chromatography with their His-Tag,the splicing reaction was carried out by mixing them in vitro.The data showed an obvious expression of three fusion proteins with expected sizes and was confirmed by His-Tag antibody directed immunoblotting.A protein band with expected vWF size was clearly observed from mixed three proteins indicating three pieces of vWF can be efficiently ligated with a dual intein-mediated protein trans-splicing.It＇s providing evidence for further study with triple vectors delivering vWF gene to eukaryotic cells by using dual inteins.

关 键 词：内含肽 蛋白质反式剪接 von WILLEBRAND因子 

分 类 号：Q5[生物学—生物化学] Q78