RACK1突变体在哺乳动物细胞中的表达及定位研究  

Expression and localization of RACK1 mutants in mammalian cell

作　　者：朱亮亮[1] 韩卢 王蓓华[1] 耿慧武[1] 潘林鑫[1] 刘晓颖[1] 范礼斌[1] 

机构地区：[1]安徽医科大学生命科学学院生物学系,合肥230032

出　　处：《安徽医科大学学报》2016年第11期1595-1599,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金青年基金(编号:81201368)

摘　　要：目的根据活化蛋白激酶C受体1(RACK1)的蛋白结构,构建其缺失突变体的真核表达质粒,研究RACK1缺失突变体在真核细胞内的表达及定位改变。方法根据RACK1蛋白的结构域特点,以pc DNA3.1-RACK1-FLAG为模板,构建真核表达质粒pc DNA3.1-RACK1(51-317)-FLAG、pc DNA3.1-RACK1(93-317)-FLAG、pc DNA3.1-RACK1(135-317)-FLAG、pc DNA3.1-RACK1(180-317)-FLAG、pc DNA3.1-RACK1(219-317)-FLAG;Western blot检测上述重组质粒在HEK 293T细胞中的表达;免疫荧光技术检测RACK1的各缺失突变体在COS7细胞中的定位情况。结果成功构建了RACK1各缺失突变体的真核表达质粒;Western blot结果表明,除pc DNA3.1-RACK1(219-317)-FLAG外,其余缺失突变体在HEK 293T细胞中均有效表达;免疫荧光实验表明RACK1缺失突变体在COS7细胞中主要定位在胞质,细胞核中也有少量分布。结论成功构建了RACK1缺失突变体真核表达质粒,并在HEK 293T和COS7细胞中成功表达;在COS7细胞中不同缺失突变体的表达定位均有差异,与野生型的RACK1相比也有所不同,表明缺失不同的结构域后其在细胞中的定位表达也发生了改变。这为研究RACK1各结构域的功能提供了重要依据。Objective To construct the eukaryotic expression plasmids of the receptor for activated C kinase 1 （ RACK1 ） deletion mutants according to the protein structure of RACK1 and observe the expression and cellular localization of RACK1 mutants in the eukaryotic cells. Methods According to the characteristics of RACK1 domains, the eukaryotic expression plasmids pcDNA3.1-RACK1 （51-317） -FLAG, pcDNA3.1-RACK1 （93-317） - FLAG, pcDNA3.1-RACK1 （ 135-317 ）-FLAG, pcDNA3.1-RACK1 （ 180-317 ）-FLAG, and pcDNA3.1-RACK1 （219-317 ）-FLAG were constructed. The expressions of RACK1 mutants plasmids in HE K 293T cells were detected by Western blot and the localizations in COS7 cells were detected by immunofluoreseence technique. Results All the plasmids of RACK1 mutants were successfully constructed. Western blot results indicated that all the mutants in HEK 293T cells expressed effectively except pcDNA3.1-RACK1 （219-317）-FLAG. Immunofluorescence experi- ments indicated that RACK1 mutants localized both in cytoplasm and nucleus, mainly in cytoplasm. Conclusion Recombinant plasmids of RACK1 mutants are constructed successfully and express effectively in HEK 293T cells and COS7 cells. However there are differences in the expression and localization of the different mutants in COS7 cells, and to contrast with the wild-type RACK1 are also similar. This situation indicates that the expression and cellular localization have some changes happened when there are some domains deficient in the RACK1 protein. The study is very important for exploring the function of the different domains of RACK1

关 键 词：突变体 质粒构建 免疫荧光 WESTERNBLOT 结构域 

分 类 号：Q28[生物学—细胞生物学] R392.7[医药卫生—免疫学]