小鼠FABP3基因全长cDNA的克隆、真核表达载体的构建及其在COS-7细胞中的表达  

Cloning of full-length cDNA of mouse FABP3 gene,construction of eukaryotic expression vector and expression of the vector in COS-7 cells

作　　者：徐力致[1] 李励芸[1] 齐秋锋[1] 闻娟[1] 陈慧梅[1] 

机构地区：[1]南京大学医学院医学遗传学教研室江苏省医学分子技术重点实验室,南京210093

出　　处：《解放军医学杂志》2010年第5期537-540,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(30800546);教育部博士点基金(200802841008)

摘　　要：目的克隆小鼠FABP3基因的全长cDNA,构建其真核表达载体,并于COS-7细胞内表达。方法利用PCR技术扩增小鼠FABP3基因的开放阅读框序列,并在3′末端连接24bp的Flag标签,克隆至真核表达载体pcDNA3.1中,构建表达质粒pcD-NA3.1-FABP3和pcDNA3.1-FABP3-Flag,并行PCR、双酶切及测序鉴定。将构建的重组质粒通过脂质体转染COS-7细胞,分别采用RT-PCR和免疫荧光法检测其FABP3mRNA及蛋白的表达情况。结果所构建的pcDNA3.1-FABP3和pcDNA3.1-FABP3-Flag重组载体可酶切出418bp的FABP3 cDNA片段和442bp的FABP3-FlagcDNA片段,经测序证实正确。转染后的COS-7细胞经RT-PCR可扩增出278bp的目的片段,且免疫荧光分析可见特异性的FABP3及Flag蛋白表达。结论构建了FABP3基因真核表达载体pcDNA3.1-FABP3和pcDNA3.1-FABP3-Flag,并于COS-7细胞中成功转录表达,为后续研究奠定了基础。Objective It had been found in previous gene expression microarray study that the gene of fatty acid binding protein 3 （FABP3） might contribute to the renal damage of patients with obesity related glomerulopathy. For further exploring the effect of FABP3 gene on podocyte lesion related obesity, the objective of present study was to construct the eukaryotic expression vector containing FABP3 gene, and to detect the expression of the vector in COS-7 cells. Methods The specific primers were designed according to the gene sequence published in GenBank, and the mouse FABP3 full-length cDNA was then cloned by RT-PCR. A 24bp FLAG coding sequence was also added to the 3＇ terminal of FABP3 cDNA using PCR. The open reading fragments of FABP3 and FABP3-flag were successfully inserted into pcDNA3.1 vector to construct eukaryotic expression vector pcDNA3.1-FABP3 and pcDNA3.1-FABP3-flag. The recombinant plasmids were transformed into the COS-7 cells for expression after the right sequence was confirmed by restriction enzymes analysis and DNA sequencing. The positive recombinants were transfected into COS-7 cells and the expression of FABP3 mRNA was detected by RT-PCR and the expression of FABP3 protein was assayed by immunofluorescence assay. Results The pcDNA3.1-FABP3 and pcDNA3.1-FABP3-flag treated with restriction enzymes EcoR Ⅰ and BamH Ⅰ produced 418bp and 442bp fragments, respectively, which were identified by sequencing as FABP3 and FABP3-flag. The object fragment of 278bp was amplified in COS-7/pcDNA3.1-FABP3 and COS-7/pcDNA3.1-FABP3-flag cells by RT-PCR. The special immunofluorescence staining was detected in the transfected cells. However, the same detections of COS-7/pcDNA3.1 cells gave negative results. Conclusion The eukaryotic expression vectors of pcDNA3.1-FABP3 and pcDNA3.1-FABP3-flag have been successfully constructed and expressed in COS-7 cells after the transfection.

关 键 词：脂肪酸结合蛋白质类 克隆 分子 遗传载体 COS细胞 

分 类 号：R349.15[医药卫生—基础医学]