Wilson病ATP7B基因铜离子结合区缺失突变体构建及功能研究

【目的】构建ATP7B基因铜离子结合区缺失突变体,研究其在Tx小鼠肝细胞中的表达及功能。【方法】利用长片断PCR和质粒重组技术构建不同铜离子结合区的缺失体,脂质体介导转染Tx小鼠肝脏细胞,然后进行高铜、低铜孵育,观察ATP7B突变蛋白在细胞内的定向移动及对铜离子转运功能的影响。【结果】ATP7B基因1-4铜离子结合区逐个缺失后,ATP7B蛋白定向移动减慢至消失;5-6铜离子结合区缺失,细胞铜转运功能停滞。【结论】ATP7B基因6个铜离子结合区功能不同,其中1-4结合区主要根据铜浓度诱导ATP7B蛋白细胞内定向移动,而5-6结合区则直接参与铜离子细胞外转运。

The Construction and Functional Analysis of the Deletion Mutant ATP7B Gene in vitro

[Objective] The deletion mutant of copper-binding domains of ATP7B gene was constructed in order to investigate its function in hepatocytes of toxic milk mouse. [ Methods] The deletion mutant expression vectors of ATP7B gene were constructed by means of long distance PCR and plas-mid-recombination. The hepatocytes of toxic milk mouse were transfected by recombinant plasmid mediated by liposome, cultured in high and low copper concentration. The trafficking of mutant ATP7B and its ability to transport copper were observed. [Results] When 1-4 copper-binding domains of ATP7B gene were deleted one by one, the trafficking of ATP7B delayed or diminished. When 5-6 copper-binding domains were deleted, copper transport stopped. [Conclusion] The function of six copper-binding domains of ATP7B gene is different. The 1-4 domains induce trafficking of ATP7B within the cell, while the 5-6 copper-binding domains are directly involved in copper transport.