Post-translational ligation and function of dual-vector transferred split CFTR gene.
- Author:
Fu-Xiang ZHU
1
;
Ze-Long LIU
;
Hui-Ge QU
;
Xiao-Yan CHI
Author Information
1. Life Science College of Ludong University, Yantai 264025, China. fuxiangmail@163.com
- Publication Type:Journal Article
- MeSH:
Animals;
Cells, Cultured;
Chlorides;
metabolism;
Codon;
genetics;
Cricetinae;
Cystic Fibrosis;
therapy;
Cystic Fibrosis Transmembrane Conductance Regulator;
genetics;
metabolism;
DNA, Complementary;
genetics;
Dependovirus;
genetics;
Genetic Therapy;
Genetic Vectors;
Humans;
Inteins;
physiology;
Kidney;
cytology;
Protein Processing, Post-Translational;
Recombinant Proteins;
genetics;
metabolism;
Trans-Splicing;
Transfection
- From:
Acta Pharmaceutica Sinica
2010;45(1):60-65
- CountryChina
- Language:Chinese
-
Abstract:
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-transfected 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.
