小鼠肝脏特异性表达载体ALB-ATP7B的构建

Construction of Mouse Liver-specific Expression Vector ALB-ATP7

[目的]构建人肝豆状核变性病ATP7B基因野生型及常见突变型的小鼠肝脏特异性表达载体ALB-ATP7B,为制备转基因小鼠及相关基因治疗作准备。[方法]定点突变法获取人群中常见的Arg778Leu及His1069Gln两种ATP7B基因突变体,定向克隆将小鼠白蛋白启动子ALB序列及野生和突变的ATP7B基因亚克隆至真核表达载体kbpala上,得到可在小鼠肝脏特异性表达的人ATP7B基因正常及突变型真核表达载体ALB-ATP7B。[结果]经测序及酶切鉴定证实,真核表达载体ALB-ATP7B构建成功。[结论]人类正常及突变ATP7B基因真核表达载体ALB-ATP7B的构建初步奠定了转基因小鼠制备的基础。

[Objective] To construct wild-type and mutant of human ATP7B gene by mouse liver-specific expression vector kbpala-albumin, which is for the preparation of transgenic mouse and further gene therapy. [Methods] Two ALB-ATP7B mutants containing the Arg778Leu and His1069Gln mutations were constructed using site-directed mutagenesis system plus site-subcloning technique. The vectors which could express wild-type and mutant of human ATP7B gene in mouse liver cells were obtained.[Results] enzyme analysis and sequencing analysis confirmed that the target genes were in right position of kbpala/albumin. [Conclusion] The construction of kbpala-alb-ATP7B gene will serve as a suitable tool for transgenic mouse in the future.