小鼠膜联蛋白A1真核表达载体的构建及其细胞内定位

目的 构建小鼠膜联蛋白A1(annexin-A1,ANXA1)真核表达载体,并检测其在NIH3T3细胞中的表达和定位.方法 采用PCR法从小鼠肝cDNA文库扩增ANXA1基因编码序列,并将其克隆至带有血凝素(HA)标记的真核表达载体pcDNA3-HA上,经PCR、酶切和测序鉴定后,将重组质粒pcDNA3-HA-ANXA1瞬时转染NIH3T3细胞,采用细胞免疫荧光法检测目的 基因表达情况.结果 菌液PCR、双酶切和DNA测序结果均表明重组质粒pcDNA3-HA-ANXA1构建正确,并可在NIH3T3细胞的胞质、胞核和胞膜中广泛表达.结论 成功构建了pcDNA3-HA-ANXA1真核表达载体,该载体中的HA-ANXA1融合基因能在哺乳动物中有效表达并正确定位,为下一步深入研究ANXAl相关的信号通路奠定了基础.

Construction of eukaryotic expression vector for mouse ANXA1 and the localization of the expression product in cells

Objective To construct the eukaryotic expression vector of mouse Annexin-A1(ANXA1),and detect its expression and loealization in NIH3T3 fibroblast cell lines. Methods The corresponding coding sequences of mouse ANXA1(GenBank accession No .AK140838)were amplified by polymerase chain reaction (PCR) from mouse liver cDNA library and then cloned into hemagglutinin(HA)tagged vector pcDNA3-HA following the routine procedures to construct a flew recombinant plasmid named pcDNA3-HA-ANXA1.After identification by double digestion with restriction endonucleases,PCR and DNA sequencing,the recombinant plasmid pcDNA3-HAANXA1 was transiently transfected into NIH3T3 cells with the Polytect transfectant reagent.The transfected cells were then cultured for a certain time, and labeled with antibody against HA and fluorescence conjugated second antibody to detect the expression and localization of HA-ANXA1 with immunocytochemical method.Results The results of identification by FCR,digestion with restriction endonucleases and DNA sequencing proved that the construction of the eukaryotic expression vector for ANXA1 was successful.After transfection into NIH 3T3 cells with the expression vector for ANXA1,it was found that the ANXA1 fusion protein was highly expressed in NIH 3T3 cells and diffusely distributed throughout the plasma membrane,the cytoplasmand the nucleus as shown by fluorescence microscopy.Conclusions The eukaryotic expression vector of HA-tagged ANXA1 was successfully constructed,which could effectively expressed in mammalian cells with a correct localization.and it could serve as an important tool in the further study of the signal pathway of ANXA1in mammalian cells.