Objective To construct prokaryotic expression vectors for three Al. DP mutants. Methods Site-directed mutagenesis was applied to wild-type ABCD1 gene cloned in the pGEX-4T-2 vector. Three mutant recombinant plasmids(pGEX-4T-2-R617C,pGEX-4T-2-P508L and pGEX-4T-2-G512S)were constructed and identified by sequencing. The expression of these mutants was detected by SDS-PAGE and western blotting. Results DNA sequencing confirmed the successful construction of three mutants. SDSPAGE and western blotting analysis showed the expression of fusion protein( NBD-GST) with 40. 5 × 103 molecular weight. Conclusion The successful construction of the three ALDP mutants would lay the foundation for further study on X-ALD molecular pathogenesis and effects of ABCD1 gene mutation on the structure and function of ALDP.%目的 构建R617C、P508L和G512S突变型表达载体并对其进行鉴定.方法 以野生型pGEX-4T-2-NBD原核表达载体为模板,通过体外诱导突变构建突变型表达载体,序列分析鉴定R617C、P508L和G512S突变重组子.结果 突变型重组载体测序结果与患者的突变位点完全符合;表达产物的12%SDS-PAGE和蛋白质印迹也显示,约40.5×103的特异性区带能被特异性单克隆抗体所识别,与预期的结果相符;通过GST蛋白纯化试剂盒获得较高纯度的目的 蛋白.结论 3个ALDP突变体的成功构建为进一步研究突变对ALDP结构和功能的影响奠定了基础,为了解X-ALD的分子发病机制创造了条件.
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