CLIC1及其点突变体与Sedlin蛋白的共定位研究

摘要

Objective To construct the eukaryotic expression plasmids of the chloride intracellular channel protein 1(CLIC1) mutants using molecular cloning techniques and observe the expression of CLIC1 mutants and their co-localization with Sedlin related to spondyloepiphyseal dysplasia tarda for further study on the cellular functions of CLIC1 protein. Methods CLIC1 mutants were amplified by PCR with the template including the full length cDNA fragment of CLIC1. Eukaryotic plasmids pcDNA3. 1-CLIC1 ( C24A)-FLAG, pcDNA3. 1-CLIC1 ( C59A)-FLAG, pcDNA3. 1-CLIC1(C24A-C59A)-FLAG were constructed respectively. The expression and localization of CLIC1 and its mutants in mammalian cells were detected by Western blot and confocal fluorescence microscopy respective-ly. Results All the plasmids of CLIC1 mutants were successfully constructed, which could be effectively expressed in HEK293T. The molecular weight of mutants was lower than the wild type. Western blot and confocal fluorescence microscopy results indicated that CLIC1 localized both in cytoplasm and nucleus, mainly in nucleus. At the same time,CLIC1 mutants localized in cytoplasm only. The mutants of CLIC1 had co-localization with CLIC1 in cyto-plasm. Conclusion Recombinant plasmids of CLIC1 mutants express effectively in eukaryotic cells. CLIC1 protein and its mutants appear to co-localize with Sedlin respectively, which implies that CLIC1 and its mutants may inter-act with Sedlin respectively.%目的：构建胞内氯离子通道蛋白1(CLIC1)点突变体的真核表达质粒，进行CLIC1突变体的表达、定位及其与迟发性脊椎骨骺发育不良病蛋白( Sedlin)的共定位研究，为进一步揭示其功能奠定基础。方法以CLIC1全长cDNA序列为模板，构建3个真核表达质粒即 pcDNA3．1-CLIC1(C24A)-FLAG、pcDNA3．1-CLIC1(C59A)-FLAG、pcDNA3．1-CLIC1( C24A-C59A)-FLAG；Western blot法检测CLIC1及其点突变体在 HEK 293T 细胞中的表达；免疫荧光技术了解CLIC1及其点突变体蛋白在COS7细胞中的定位及与Sedlin的共定位情况。结果成功构建了CLIC1点突变体的真核表达质粒；Western blot 结果显示3个点突变体蛋白在HEK293T细胞中均能表达，突变体的分子量低于野生型的；免疫荧光实验表明CLIC1蛋白在细胞质和细胞核都有表达，但主要在细胞核。 CLIC1点突变体蛋白定位在胞质中，细胞核中无分布，与 Sedlin 蛋白在胞质存在共定位。结论CLIC1的3种点突变体均能在哺乳动物细胞中有效表达；CLIC1及其点突变体与Sedlin蛋白均存在共定位，提示两种蛋白之间可能存在相互作用。