高效表达型T克隆载体的构建及其特性

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peDNA3，回收线性化质粒片段与三磷酸胸腺嘧啶脱氧核苷酸（driP）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72h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cwIFINA5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。

Characterization of a highly efficient expression T-vector

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.