Cloning of CGI-100 gene and reconstruction of its eukaryotic expressive vector / 重庆医科大学学报

ABSTRACT

Objective:To clone human CGI-100 gene and reconstruct its eukaryotic expressive vector for further investigation.Methods:The full coding domain sequence of human CGI-100 gene was cloned from human leukemic K562 cells with RT-PCR and was sub-cloned into pMD18-T Simple vector.After confirmed by DNA sequencing,the targeted DNA fragment,digested with BamHⅠand PstⅠ,was directionally cloned into eukaryotic expressive plasmid pIRES2-EGFP,then,the reconstructed plasmid was identified with enzyme digestion,PCR and sequencing.Results:It was demonstrated that the full coding domain sequence of human CGI-100 gene was accurately cloned into digestion sites between BamHⅠand PstⅠin pIRES2-EGFP without mutation and transposition.Conclusion:The reconstruction and verifying of eukaryotic expressive plasmid containing CGI-100 gene are successful,which establishes the foundation of further investigation.