小鼠子宫珠蛋白结合蛋白真核表达载体构建及其在COS-1细胞的表达
| 作者: |
1李晨,
2史小娟,
2黄艳红,
2冯丹丹,
2罗自强
1 中南大学湘雅医学院生理学系,长沙 410078; 长治医学院生理学教研室,山西 长治046000 2 中南大学湘雅医学院生理学系,长沙 410078; |
| 通讯: |
罗自强
Email: luozq1962@163.com |
| DOI: | 10.3978/j.issn.2095-6959.2013.01.003 |
摘要
构建小鼠子宫珠蛋白结合蛋白(mouse uteroglobin binding protein, mUGBP)真核表达载体,观察其在COS-1细胞中的表达和定位。方法: 用RT-PCR方法扩增mUGBP的cDNA序列,将其插入pEGFP-N1载体的多克隆位点构建pEGFP-UGBP重组质粒。采用脂质体转染法将重组质粒转染到无内源性mUGBP表达的非洲绿猴肾成纤维细胞株COS-1中,观察其在细胞内的表达,并用Western免疫印迹方法进行验证。结果: pEGFP-UGBP重组质粒经酶切鉴定以及测序证实,插入的目的基因序列完全正确。激光共聚焦显微镜观察发现,转染了pEGFP-UGBP重组质粒的细胞株可见绿色萤光蛋白的表达;Western免疫印迹检测发现细胞膜组分中可检获上述转染了重组质粒的细胞所表达的融合蛋白。结论: 成功构建了pEGFP-UGBP重组质粒并进行了真核表达。
关键词：
Construction of mouse uteroglobin binding protein eukaryotic expression vector and its expression in COS-1 cells
CorrespondingAuthor: LUO Ziqiang Email: luozq1962@163.com
DOI: 10.3978/j.issn.2095-6959.2013.01.003
Abstract
To construct a eukaryotic vector of mouse uteroglobin binding protein (mUGBP) and to detect its expression and localization in COS-1 cells. Methods: The cDNA sequences of mUGBP were amplified by RTPCR, and then they were cloned into the pEGFP-N1 vector to construct a new recombinant plasmid pEGFPUGBP. The recombinant plasmid was subsequently transfected into COS-1 cells with liposome transfection reagent. The expression and localization of the mUGBP-GFP fusion protein were analyzed by confocal microscope and Western blot. Results: Double digestion by restriction endonuclease and DNA sequencing demonstrated that the recombinant plasmid pEGFP-UGBP was correctly constructed. Under the confocal microscope, fusion protein autofluorescence was found in COS-1 cells transfected with the recombinant plasmid pEGFP-UGBP. The expression of the fusion protein was detected by Western blot analysis in membrane fractions of transfected COS-1 cells. Conclusion: The recombinant vector pEGFP-UGBP is constructed and effectively expressed in mammalian cells.