用同源重组法修饰含人α类珠蛋白基因簇的细菌人工染色体

The Modification of a Bacterial Artifical Chromosome Containing Human α-globin Gene Cluster by Homologous Recombination

采用RecA蛋白介导的同源重组的方法 ,对本实验室筛选出的包含完整人α 类珠蛋白基因簇的细菌人工染色体 (BAC)DNA进行删除修饰。首先采用PCR的方法 ,分别在预删除的HS - 40增强子区域的上游和下游克隆长度约为 5 0 0bp的两段同源序列 ,并一起克隆到构建载体 pBV的XbaI和HindIII位点上 ,SalI酶切后回收 1kb左右的片段并克隆到温度敏感的穿梭质粒 pSV -RecA中 ,转化带有人α 类珠蛋白基因簇的BACDNA的感受态大肠杆菌DH10B ,经氯霉素正筛选和镰孢菌酸的负筛选 ,获得发生两次同源重组后只含BACDNA而穿梭载体已丢失的菌株 ,经Southern杂交鉴定 ,获得了HS - 40被定位删除的BAC突变体克隆。

By RecA protein mediated homologous recombination method, we successfully deleted the HS-40 fragment from a bacterial artificial chromosome containing complete human α-globin gene cluster screened by our lab. Two mutant boxes(A and B) fragments located at the two terminals of HS-40 were cloned into the Xba Ⅰ and Hind III sites of pBV building vector, then the 1.1kb A+B fragment was recovered from the building vector and inserted into the Sal Ⅰ site of the shuttle vector pSV-RecA, competent DH10B E. Coli containing BAC DNA was tranformed by the shuttle vector,after chloramphenicol positive selection and fusatic acid negative selection,two times of homologous recombination happened and the HS-40 fragment was successfully deleted from the BAC DNA which is characterized by Southern blot,suggested that this method could modified BAC DNA accurately containing high percentage of repeat sequence.