论文部分内容阅读
目的: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。
OBJECTIVE: To construct a mouse eukaryotic expression vector for mouse uteroglobin binding protein (mUGBP) and to observe its expression and localization in COS-1 cells. Methods: The cDNA sequence of mUGBP was amplified by RT-PCR and inserted into the multiple cloning site of pEGFP-N1 vector to construct pEGFP-UGBP recombinant plasmid. The recombinant plasmids were transfected into COS-1 cells of African green monkey kidney fibroblast without endogenous mUGBP expression by lipofectamine. The expression of the recombinant plasmids was observed in the cells and verified by Western immunoblotting. Results: The recombinant plasmid pEGFP-UGBP confirmed by restriction analysis and sequencing confirmed that the insertion of the target gene sequence is correct. Laser confocal microscopy showed that the expression of green fluorescent protein was observed in the cell lines transfected with the recombinant plasmid of pEGFP-UGBP. Western blotting showed that the fusion protein expressed in the cell membrane fractions transfected with the recombinant plasmids . Conclusion: The recombinant plasmid pEGFP-UGBP was successfully constructed and eukaryotic expressed.