内含肽介导的氯离子通道蛋白CFTR的反式剪接

研究利用内含肽(intein)的蛋白质反式剪接功能在大肠杆菌中对囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane regulator, CFTR)的反式剪接作用.CFTR基因突变导致一种常染色体隐性遗传疾病囊性纤维化(cystic fibrosis, CF).将CFTR的cDNA于剪接反应所需的保守性氨基酸残基Ser-660前断裂为N端和C端，分别与split mini Ssp DnaB 内含肽的106个氨基酸残基的N端和48个氨基酸残基的C端编码序列融合，构建到原核表达载体pBV220 诱导表达后SDS-PAGE可见预期大小剪接形成的CFTR蛋白条带，Western印迹用CFTR特异性抗体进一步证明为剪接所产生的CFTR蛋白，表明内含肽可有效催化CFTR的反式剪接.

Intein-mediated Trans-splicing of Chloride Ion Channel CFTR

By exploring intein-catalyzed protein splicing we investigated the trans-splicing of cystic fibrosis transmembrane regulator (CFTR) in E. coli.The mutations of CFTR gene lead to cystic fibrosis(CF), an autosomal recessive disorder. The CFTR cDNA was broken into two halves of N-part and C-part before Ser660 conserved for protein splicing, then fused to 106 and 48 amino acid residues containing N- and C- termini coding sequences of split mini Ssp DnaB intein, respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220.Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of CFTR protein produced by splicing on SDS-PAGE. Western blotting via CFTR-specific antibodies further confirmed that this protein band was trans-spliced