【摘要】 目的:探索Mpl与绿色荧光蛋白GFP基因共同转染哺乳动物细胞NIH3T3的方法。方法:采用PCR方法将GFP基因与Mpl基因构建融合荧光蛋白的真核表达载体,用脂质体介导转染NIH3T3细胞和筛选稳定细胞系,使用荧光显微镜方法和Western blotting检测转染效果。结果:利用PCR方法有效扩增了Mpl基因,构建了融合荧光蛋白的真核表达载体,序列分析表明所构建的含Mpl基因的质粒与设计相同,使用荧光显微镜方法和Western blotting检测Mpl融合绿色荧光蛋白表达载体成功转染NIH3T3细胞。结论:成功构建了Mpl荧光表达载体,融合基因可以在NIH3T3细胞中稳定表达,为进一步研究Mpl的生物学活性及其与hNUDC蛋白相互作用提供了重要的理论依据。更多还原

【Abstract】 Objective: To construct eukaryotic expression vector of Mpl and express green fluorescent (GFP) fusion gene in mammalian cells NIH3T3. Methods: GFP-Mpl fusion gene was synthesized by PCR strategy, and the sequence was inserted into cells with cationicliposome mediated by gene transfection technique. The result of the construction and expression was determined by fluorescencemicroscope and Western blotting assay. Results: The human Mpl gene was effectively amplified by PCR. The expression vector was constructed, and the result of the DNA sequencing showed that the constructed plasmid containing the Mpl gene. The result of fluorescence microscope and Western blotting analysis showed Mpl-GFP was expressed in NIH 3T3 cells successfully. Conclusions: The construction of the eukaryotic expression vector of Mpl and GFP fusion protein has been achieved, and the Mp1-GFP fusion protein was expressed very well in NIH3T3 cells. It has founded an important basis for studying the activity of the Mpl and the interaction between Mp1 and hNUDC protein.更多还原