Mpl与绿色荧光蛋白融合基因的真核载体构建及表达  

Construction and Expression of Eukaryotic Expression Vector of Mpl and Green Fluorescent Protein Fusion Gene

作　　者：陈彦球[1] 张裕平[2] 徐培林[2] 杨燕[3] 

机构地区：[1]中山大学第一附属医院,广东广州510008 [2]中山大学教育部基因工程重点实验室,广东广州510275 [3]中山大学公共卫生学院,广东广州510008

出　　处：《现代生物医学进展》2009年第9期1612-1615,共4页Progress in Modern Biomedicine

基　　金：广东省自然科学基金博士启动资助项目(06300645);教育部博士点基金新教师资助项目(20070558275);中山大学2007实验室开放基金项目(KF200707)

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。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 fl croscope 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 Mp 1-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 Mp 1 and hNUDC protein.

关 键 词：MPL GFP NIH3T3细胞 

分 类 号：R392.12[医药卫生—免疫学]