髓样相关蛋白8真核表达载体的构建及其细胞内定位  

Construction and localization in cells of eukaryotic expression vector for myeloid-related protein-8

作　　者：伍丽琼[1] 吴翠玲[1] 王娟[1] 徐佳[1] 邓鹏[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室,广东省蛋白质组学重点实验室,广州市510515

出　　处：《实用医学杂志》2010年第6期907-909,共3页The Journal of Practical Medicine

基　　金：国家自然科学基金项目(编号:30670828);国家重点基础研究发展计划(973计划)(编号:2002CB13005);国家自然科学基金委员会-广东省人民政府自然科学联合基金重点项目(编号:U0632004)

摘　　要：目的:构建小鼠髓样相关蛋白8(MRP8)的真核表达载体,观察其在NIH3T3细胞中的表达定位情况。方法:提取BALB/c小鼠肝脏组织的总RNA,通过逆转录-聚合酶链反应扩增得到MRP8编码序列,并将其克隆到带有血凝素(HA)标记的载体pcDNA3-HA上,随后将重组质粒瞬时转染NIH3T3细胞,利用荧光显微镜观察结果。结果:重组质粒经聚合酶链反应、酶切和测序鉴定证明构建正确,并且该质粒能够在NIH3T3细胞的胞浆、胞核中广泛表达,表达产物主要定位在细胞核中。结论:成功构建带有HA标签的MRP8真核表达载体。该载体能在哺乳动物细胞中有效表达并正确定位,为进一步研究MRP8作用细胞的信号通路提供了一个重要的工具。Objective To construct eukatyotic expression vector for myeloid-related protein-8 （MRP8）, and to detect the localization of MRP8 in NIH3T3 cells. Methods Total RNA from the hepatic tissue of BALB/c mice was extracted,and the corresponding coding sequences of MRP8 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）and cloned into hemagglutinin （ HA ）-tagged mammalian cell expression vector pcDNA3-HA.The recombinant plasmid pcDNA3-HA-MRP8 was transfected into NIH3T3 ceils, which were observed under fluorescence microscope. Results The recombinant plasmid was verified by polymerase chain reaction （PCR）, enzyme digestion and sequence analysis. Under fluorescence microscopy, the fusion protein was highly expressed in the cytoplasm and nucleus of a wide range of NIH3T3 cells, especially in the nucleus. Conclusion The MRP8-HA expression vector has been successfully constructed, with an effective expression and localization in mammalian cells, which provides an important tool for the study on the signaling pathway induced by MRP8.

关 键 词：髓样相关蛋白8 血凝素类 转染 细胞内定位 载体构建 

分 类 号：R346[医药卫生—基础医学]