肝脏特异性表达载体Kbpala/Alb-ATP7B的构建及表达

Construction and expression of mouse liver-specific expression vector Kbpala/Alb-STBXATP7B

目的:构建小鼠白蛋白启动子引导下携带野生及常见突变型Arg778Leu、His1069Gln的人ATP7BcD-NA的肝脏特异性表达载体Kbpala/Alb-ATP7B,并检测其表达情况。方法:定点突变法获取人群中常见的Arg778Leu及His1069Gln两种ATP7B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ATP7BcDNA依次亚克隆至转基因载体Kbpala上,得到可在肝脏特异性表达的正常及突变型人ATP7B的转基因载体Kbpala/Alb-ATP7B。将其短暂转染BRL细胞及BHK细胞,通过Western blotting检测其蛋白表达情况。结果:经酶切鉴定及定点测序证实,转基因载体Kbpala/Alb-ATP7B构建成功;Western blotting显示仅在肝脏细胞实现表达。结论:带有人类ATP7BcDNA的野生及常见致病突变型的转基因载体Kbpala/Alb-ATP7B构建成功并在肝脏细胞特异性表达。

AIM： To construct the liver- specific transgene vectors encoding wildtype as well as most commnon disease mutant ATPTB eDNA under the control of mouse albumin promoter and to explore their expression. METHODS： Two Kbpala/ Alb- ATP7B mutants containing the Arg778Leu and His1069Gln mutations were constructed using site- directed mutagenesis system plus site - subcloning technique. The vectors expressed wild- type and mutants of human ATP7B in mouse liver cells were obtained and transiently transfected into BRL and BHK cell lines. Western blotting analysis was utilized to detect the expression of human ATP7B. RESULTS： Enzyme analysis and sequencing analysis confirmed that the target genes were ATP7B and in right position. The results of Western blotting showed that the gene products were expressed specifically in liver cells. CONCCONCLUSION： The Kbpala/Alb - ATP7B vectors were constructed successfully and the liver- specific expression of human ATP7B proteins were conformed.