遗传 ›› 2001, Vol. 23 ›› Issue (3): 187-191.

• 论文 • 上一篇    下一篇

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

冯东晓;刘德培;黄粤;梁植权 FENG Dong-xiao;LIU De-pei;HUANG Yue;LIANG Zhi-quan   

  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 National Laboratory of Medical Molecular Biology,Institute of Basic Me dical Sciences,CAMS and PVMC,Beijing,100005,China
  • 收稿日期:1900-01-01 出版日期:2001-06-10 发布日期:2001-06-10

The Modification of a Bacterial Artifical Chromosome Containing

  • Received:1900-01-01 Online:2001-06-10 Published:2001-06-10

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.

关键词: homologous recomb ination, α-珠蛋白 Keywords, bacterial artificial chromosome, 同源重组, 细菌人工染色体