基于蛋白质剪接的BHK细胞Ser^(660)前断裂的CFTR基因转移  

Protein Splicing Based Gene Transfer of Split Cystic Fibrosis Transmembrane Conductance Regulator Severed Before Ser^(660) in BHK Cells

作　　者：朱甫祥[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,山东烟台264025

出　　处：《中国生物化学与分子生物学报》2010年第5期429-435,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：山东省自然科学基金(No.Y2005D14);烟台市科技发展计划(No.2008152);教育部留学回国人员科研启动基金(No.20071108);鲁东大学学科建设经费资助项目~~

摘　　要：利用内含肽(intein)的蛋白质反式剪接技术,研究双载体真核细胞转囊性纤维化跨膜电导调节体(CFTR)基因,通过翻译后连接成为完整的功能性CFTR蛋白.应用基因重组技术,将人CFTRcDNA于剪接反应所需保守残基Ser660前断裂为N端和C端两部分,分别与split Ssp DnaB intein编码序列融合,构建到真核表达载体pEGFP-N1和pEYFP-N1.用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),48h后Western印迹观察CFTR蛋白质的连接,并用全细胞和单通道膜片钳技术记录Cl-通道电流.基因共转染细胞可观察到明显的由蛋白质反式剪接形成的完整CFTR蛋白,膜片钳记录到较高的全细胞Cl-电流和与转野生型CFTR基因细胞相似的单Cl-通道开放活性,提示CFTR功能的恢复.内含肽可作为一种技术策略用于双载体转CFTR基因,为应用双腺相关病毒载体(AAV)转基因的囊性纤维化疾病(CF)基因治疗提供了依据.A dual-vector system was used to transfer cystic fibrosis transmembrane conductance regulator （CFTR） gene into eukaryotic cells to produce the functional protein through post-translational ligation. The coding sequences of split Ssp DnaB intein were fused with each of the human CFTR N-and C-half severed at Ser660,and then inserted into eukaryotic expression vector pEGFP-N1 and pEYFP-N1, respectively. Forty eight hours after cotransfection of the two vectors into baby hamster kidney （ BHK） cells,the ligated CFTR from two polypeptide fragments was observed by Western blotting. A high chloride current was recorded by whole-cell and single channel patch clamps indicating a similar activity to that of the wild type CFTR. Our results demonstrated that the intein-based strategy could be used to recover the CFTR gene function with two vectors,and might be applicable in the treatment of cystic fibrosis by using a dual adeno-associated virus （AAV） vector system.

关 键 词：蛋白质反式剪接 囊性纤维化跨膜电导调节体 基因转移 膜片钳 

分 类 号：Q73[生物学—分子生物学]