未知基因CGI-100的克隆及其真核表达载体的构建

Cloning of CGI-100 gene and reconstruction of its eukaryotic expressive vector

目的:为探讨研究未知基因CGI-100的功能,克隆人CGI-100基因并构建其真核表达载体。方法:通过RT-PCR从人白血病K562细胞中克隆CGI-100基因全长编码区,将该cDNA片段亚克隆至载体pMD18-T SimpleT中,经测序鉴定后,用BamHⅠ和PstⅠ双酶切将目的cDNA片段定向克隆至相同处理的pIRES2-EGFP真核表达质粒中,然后经酶切、PCR鉴定和DNA序列测定三重鉴定方法对重组质粒进行鉴定。结果:经酶切、PCR及测序鉴定证实,CGI-100基因全长编码区cDNA被准确正向插入至pIRES2-EGFP质粒的酶切位点BamHⅠ和PstⅠ之间,未发现碱基突变或移位。结论:成功地构建并鉴定了含CGI-100基因全长编码区的真核表达质粒,为以后研究该基因的功能奠定了基础。

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 BamH Ⅰ and Pst Ⅰ ,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 BamH Ⅰ and Pst Ⅰ in 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.