遗传 ›› 2001, Vol. 23 ›› Issue (3): 187-191.
冯东晓;刘德培;黄粤;梁植权 FENG Dong-xiao;LIU De-pei;HUANG Yue;LIANG Zhi-quan
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500bp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。表明同源重组的方法可以对含有较多重复序列的BACDNA进行准确的删除修饰。
Abstract:By RecA protein mediated homologous recombination method, we successfully delet ed 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) fra gments located at the two terminals of HS-40 were cloned into the XbaⅠ and HindIII sites of pBV building vector, then the 1.1kb A+B fragment was recove red from the building vector and inserted into the SalⅠ site of the shuttle vector pSV-RecA, competent DH10B E. Coli containing BAC DNA was tranformed by t he shuttle vector,after chloramphenicol positive selection and fusatic acid neg ative selection,two times of homologous recombination happened and the HS-40 fra gment was successfully deleted from the BAC DNA which is characterized by Southe rn blot,suggested that this method could modified BAC DNA accurately containing high percentage of repeat sequence.