Wilson蛋白N端多克隆抗体的制备和纯化

【目的】制备并纯化Wilson蛋白(ATP7B)的N端多克隆抗体,为进一步研究其基因表达蛋白的结构和功能打下基础。【方法】RT-PCR扩增目的基因,GST基因融合载体表达融合蛋白,亲和层析进行蛋白纯化,Thrombin酶切并收集目的蛋白,免疫家兔,ELISA检测抗体滴度,离子交换层析纯化抗体血清,Westernblot检测抗体特异性。【结果】ELISA方法检测抗体滴度达到了1∶2500,Westernblot表明该抗体有很好的特异性,非变性SDS-PAGE显示纯化后的抗体IgG纯度很高。【结论】应用该方法成功制备了Wilson病蛋白质量的多克隆抗体。

Wilson蛋白离子结合功能的研究

目的 研究Wilson蛋白 (ATP7B酶 )的离子结合功能 ,探讨锌剂治疗Wilson病的机制。方法 应用逆转录 聚合酶链反应 (RT PCR)扩增目的基因 ,GST基因融合载体表达并纯化融合蛋白 ,凝血酶酶切并收集目的蛋白 ,应用离子螯合层析和放射性锌印迹技术研究蛋白不同离子的结合能力。结果 ATP7B的离子结合能力为Cu(Ⅰ ) >Zn(Ⅱ ) >Ni(Ⅱ ) >Cu(Ⅱ ) ,并发现锌离子和铜离子对蛋白的结合具有竞争作用。结论 本实验证明了ATP7B以一价铜离子的形式行使其运铜功能 ,ATP7B不仅有结合铜离子的功能 ,而且有结合其他离子的功能 ;铜锌两种金属离子在蛋白结合位点的竞争作用可能是锌剂治疗WD的机制之一。

Analysis of the human Atox 1 homologue in Wilson patients

AIM: To analyze the metallochaperone antioxidant-1 (Atox1) gene sequence in Wilson disease patients. METHODS: Mutation analysis of the four exons of the Atox1 gene including the intron- exon boundaries was performed in 63 Wilson disease patients by direct sequencing. RESULTS: From 63 selected patients no mutations were identified after the entire coding region including the intron- exon boundaries of Atox1 were sequenced. One known polymorphism within the Atox1 gene (5’UTR -99 T>C) in 31 (49%) of the Wilson patients as well as one previously undescribed variation (5’UTR -68 C>T) in 2 of the Wilson patients could be detected. Statistical analyses revealed that the existence of a variation within the Atox1- gene showed a tendency towards an earlier onset of the disease. CONCLUSION: Based on the data of this study, no major role can be attributed to Atox1 in the pathophysiology or clinical variation of Wilson disease.