TMD2前断裂CFTR翻译后的连接及氯离子通道功能

Post-translational ligation of split CFTR severed before TMD2 and its chloride channel function

摘要:

CFTR基因突变导致一种常染色体隐性遗传疾病——囊性纤维化 (CF)。利用split Ssp DnaB intein的蛋白质反式剪接技术的真核细胞双载体转CFTR基因，旨在研究翻译后水平CFTR的连接，以及由其建立的氯离子通道功能。于CFTR膜内第2个跨膜结构域 (TMD2) 前的Glu838密码子后将其cDNA断裂为N端和C端两部分，与具有蛋白质反式剪接作用的split Ssp DnaB intein编码序列融合，分别插入到载体pEGFP-N1和pEYFP-N1，构建一对真核表达载体pEGFP-NInt和pEYFP-IntC。用脂质体将这对载体共转染至幼年仓鼠肾细胞 (BHK)，瞬时表达实验用Western blotting观察CFTR蛋白质的连接，并用膜片钳技术记录Cl?通道电流。结果显示，基因共转染细胞呈现完整的CFTR蛋白条带，膜片钳记录到全细胞Cl?电流和单个Cl?通道开放活性。结果表明split Ssp DnaB intein的蛋白质反式剪接技术可用于双载体共转移CFTR基因，为CF基因治疗应用双腺相关病毒载体 (AAV) 转运CFTR基因，克服AAV的容量限制提供了依据。

Abstract:

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These dada demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.