高效表达型T克隆载体的构建及其特性  

Characterization of a highly efficient expression T-vector

作　　者：卢银平[1] 祝建芳[1] 刘朝[1] 董继华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院病毒研究室,湖北武汉430022

出　　处：《中国实验诊断学》2009年第9期1137-1139,共3页Chinese Journal of Laboratory Diagnosis

基　　金：教育部高等院校博士点基金(20070487152);湖北省自然科学基金(2008CDB188)

摘　　要：目的研制高效表达型T载体用于基因的表达及蛋白功能分析。方法限制性内切酶EcoR V酶切真核表达载体pcDNA3,回收线性化质粒片段与三磷酸胸腺嘧啶脱氧核苷酸(dTTP)70℃退火,构成T克隆载体并回收纯化。将增强绿色荧光蛋白基因(EGFP)和中国旱獭α干扰素基因(cwIFNA5)分别扩增后直接克隆到新构建的表达型T载体,转化感受态细菌,挑选阳性菌并制备质粒瞬时转染真核细胞,荧光显微镜观察EGFP的表达,病毒保护试验检测中国旱獭α干扰素的生物学活性。结果成功构建了表达型T载体,外源基因EGFP和cwIFNA5克隆进该载体后可以进行高效表达,基因转染72 h后,荧光显微镜观察到EGFP基因转染细胞荧光阳性率在80%以上,荧光强度高;病毒保护试验表明cwIFNA5基因转染细胞培养上清干扰素效价达1∶640。结论新构建的表达型T载体可用于基因的克隆之外,还可以直接用于基因的高效表达及蛋白质功能分析。Objective To develop a highly efficient expression T-vector to clone and express target gene. Methods PeDNA3 plasmids were digested with restriction enzyme EcoR V, the 3＇ terminal of the linearized plasmids was added a single deoxythymidine （dTIP） by annealing reaction. Enhanced green fluorescent protein gene （EGFP） and Chinese marmot interferon alpha gene （cwIFNAS） were cloned into this T-vector after gene amplification, EGFP and cwIFNA5 were expressed in Siha cells and BHK cells, respectively. Results The expression T-vector was constructed successfully. EGFP and cwlFNA5 were expressed efficiently, and IFN- α possessed high biological activity. Conclusion This expression T-vector not only could be used to clone target gene, but also to highly efficient repress target gene.

关 键 词：表达型T载体 基因克隆 基因表达 

分 类 号：R346[医药卫生—基础医学]