中文摘要：

目的运用分子克隆技术构建活化蛋白激酶C受体1（RACK1）突变体的真核表达质粒,进行RACK1突变体的表达、定位及其与胞内氯离子通道蛋白1（CLIC1）蛋白的共定位研究,为其进一步的功能研究奠定基础。方法以RACK1全长c DNA序列为模板,构建真核表达质粒pc DNA3.1-RACK1-N（1-138）-FLAG、pc DNA3.1-RACK1-C（130-317）-FLAG;Western blot法检测突变体在哺乳动物细胞中的表达;用免疫荧光技术了解突变体蛋白在哺乳动物细胞中的共定位情况。结果成功构建了RACK1突变体的真核表达质粒;Western blot法检测到蛋白主要在胞质中表达,核内有部分表达;免疫荧光实验表明,RACK1及突变体蛋白主要定位在胞质与核膜,细胞核中也有少量表达,与CLIC1蛋白存在共定位。结论成功构建了RACK1缺失突变体,并在真核细胞中成功表达;RACK1及突变体与CLIC1蛋白在哺乳动物细胞中均存在不同程度的共定位,蛋白之间可能存在相互作用,为RACK1蛋白的功能研究奠定了基础。

英文摘要：

Objective To construct the eukaryotic expression plasmids of the receptor for activated C kinase 1（ RACK1） mutants using molecular cloning techniques and observe the expression and cellular localization of RACK1 mutants and their co-localization with chloride intracellular channel protein 1（ CLIC1） for further study on the cellular functions of RACK1 protein. Methods RACK1 mutants were amplified by PCR with the template including the full length c DNA fragment of RACK1. Eukaryotic plasmids pc DNA3. 1-RACK1-N（ 1-138）-FLAG,pc DNA3. 1-RACK1-C（ 130- 317）-FLAG were constructed respectively. The cellular expression and localization of RACK1 and its mutants in mammalian cells were detected by Western blot and confocal fluorescence microscopy respectively. Results All the plasmids of RACK1 mutants were successfully constructed. Western blot and confocal fluorescence microscopy results indicated that RACK1 and its mutants localized both in cytoplasm and nucleus,mainly in cytoplasm. The co-localization existed between RACK1 and CLIC1,and the mutants of RACK1 had similar co-localization with CLIC1. Conclusion Recombinant plasmids of RACK1 mutants are constructed successfully and express effectively in eukaryotic cells. RACK1 protein and its mutants appear to co-localize with CLIC1 respectively,which implies that RACK1 and its mutants may interact with CLIC1 respectively in mammalian cells. The study is very important for exploring the function of RACK1.