改进方案构建人糖皮质激素受体α基因正、反义重组逆病毒载体

Construction of Retroviral Recombinant Vectors Carrying Sense- and Antisense-hGRα cDNA by an Improved Ligasing Protocol

目的 构建人糖皮质激素α(hGRα)cDNA的表达重组体 ,为较大DNA片段的克隆提供借鉴 ,并为利用反义核酸技术探讨hGRα功能及其作用机制提供实验材料。方法 L -PCR扩增hGRαcDNA ,克隆至T载体 ;随后通过改进的连接方案进行亚克隆。结果 限制性酶增和序列分析表明克隆及亚克隆成功。结论 成功构建于hGRαcDNA的逆转录病毒载体pLXSN正义及反义重组子。

Objective To construct the recombinant expression vector carrying human glucocorticoid receptor α(hGRα) cDNA to provide experiences for cloning of large DNA fragments and offer experimental materials for studying the function and medchanism of hGRα by antisense nucleic acid technique. Methods hGRα cDNA was amplified from an expressional phage library of human white cells by L-PCR, and cloned into pGEM-Teasy vector. Subsequently, the hGRα cDNA fragment digested from the recombinant T vector was sub-cloned into retroviral vector pLXSN in sense and antisense orientations, respectively through an improved ligasing protocol. Results Restriction endonuclease analysis and sequencing showed that cloning and sub-cloning of hGRα were successful. Conclusion Retroviral recombinant vectors carrying sense-and antisense-hGRα cDNA were successfully constructed.