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

作　　者：冯东晓[1] 刘德培[1] 黄粤[1] 梁植权[1] 

机构地区：[1]中国医学科学院基础医学研究所中国协和医科大学基础医学院医学分子生物学国家重点实验室,北京100005

出　　处：《遗传》2001年第3期187-191,共5页Hereditas（Beijing)

基　　金：国家自然科学基金!重大项目 (No .3 9893 3 2 0 )资助

摘　　要：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.

关 键 词：细菌人工染色体 同源重组 Α-珠蛋白 修饰 

分 类 号：Q78[生物学—分子生物学]