双载体断裂CFTR基因转移及翻译后的连接和功能

Post-translational ligation and function of dual-vector transferred split CFTR gene

囊性纤维化跨膜电导调节体(CFTR)基因突变导致一种常染色体隐性遗传病囊性纤维化(CF),利用腺辅助病毒(AAV)载体转运CFTR基因的基因疗法受到AAV载体容量的限制。本文采用intein的蛋白质反式剪接技术,以双载体转运CFTR基因,研究了于其调节结构域(R)断裂成两部分的CFTR基因翻译后的连接及其产生的Cl-通道功能。将人CFTRcDNA于R结构域的Ser712密码子前断裂,构建一对融合SspDna Bintein编码序列的真核表达载体。将这对载体共转染培养的幼年仓鼠肾(BHK)细胞,通过瞬时表达,全细胞和单通道膜片钳记录Cl-电流,并用Westernblotting观察CFTR蛋白的剪接。结果表明,共转染细胞显示较高的全细胞Cl-电流和单个Cl-通道开放活性,说明CFTR的Cl-通道功能的恢复,用CFTR特异性抗体进行的细胞总蛋白Westernblotting显示有完整的CFTR蛋白条带形成,表明intein可有效连接翻译后的两部分CFTR蛋白。结果提示,蛋白质剪接技术可有效用于双载体系统转运CFTR基因,为进一步应用双AAV载体转运CFTR基因的CF基因治疗研究提供了实验依据。

The mutation of cystic fibrosis transmembrane conductance regulator （CFTR） gene leads to an autosomal recessive genetic disorder cystic fibrosis （CF）. The gene therapy for CF using adeno-associated virus （AAV） vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein- mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney （BHK） cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-tansfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors,encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.