摘要
目的:构建携带绿色荧光蛋白(GFP)报告基因及PAX6基因的重组真核表达载体,观察其在人胚肾细胞(293FT)细胞中的表达。方法:采用聚合酶链式反应(PCR)从大鼠脑组织中获取PAX6基因,经连接T载体测序验证正确后,与真核表达载体p EF1α-IRES-Ac GFP经Sal I/Bam HI双酶切后;经T4 DNA连接酶连接,获得重组质粒p EF1α-PAX6-IRES-Ac GFP,用菌液PCR、Sal I/Bam HI双酶切及测序鉴定正确后,用脂质体法转染293FT细胞,采用倒置荧光显微镜观察GFP的表达、蛋白印迹(Western blot)法检测PAX6蛋白表达。结果:重组质粒p EF1α-PAX6-IRES-Ac GFP经RT-PCR和双酶切均得到大小为1 269 bp的目的条带,测序鉴定该序列与Gen Bank中大鼠PAX6基因序列的同源性达100%,插入基因的大小和方向正确;重组质粒转染293FT细胞后可见GFP绿色荧光,Western blot显示PAX6蛋白表达。结论:成功构建了PAX6真核表达重组质粒p EF1α-PAX6-IRES-Ac GFP,能在293FT细胞中表达PAX6蛋白。
Objective： To construct a green fluorescence protein report gene（ GFP） and an eukaryotic expression vector of rat PAX6 gene and,observe its expression in 293 FT cells. Methods： The rat PAX6 gene was amplified by Polymerase Chain Reaction（ PCR）. After verified correctness of sequencing by connection with T vector,the recombinant products of the p EF1α-PAX6-IRES-Ac GFP were gained by T4 ligase connecting p UCm-T-PAX6 and eukaryotic expression vector p EF1α-IRES-Ac GFP with restriction enzymes SalⅠ / Bam H. After identified by colony PCR and sequencing,p EF1α-PAX6-IRES-Ac GFP was transfected into 293 FT cells with Lipofectamine2000 assay. The expression of PAX6 protein and GFP were detected by western blot and fluorescence microscope. Results： A specific band of 1269 bp was detected from recombinant plasmid p EF1α-PAX6-IRES-Ac GFP by digestion of SalⅠand Bam HⅠand RT-PCR. Sequencing and identification showed that homology between this sequence and the rat PAX6 gene sequence from Gen Bank was 100%,and the size and the direction of the inserted gene were right. After transfection,293 FT cells showed green fluorescence under fluorescence microscope. A band of PAX6 protein from cells was detected by western blot assay. Conclusion： The eukaryotic expression vector of PAX6 is constructed successfully and can obtain high PAX6 protein expression in 293 FT cells.
出处
《贵阳医学院学报》
CAS
2016年第3期288-293,共6页
Journal of Guiyang Medical College
基金
贵州省科技厅基金[黔科合J字(2015)2011号]
贵州省优秀科技教育人才省长专项资金项目[黔省专合字(2012)41号]
关键词
绿色荧光蛋白
PAX6基因
表达载体
真核
脂质体
转染
green fluorescence protein
PAX6 gene
expression vector
eukaryotic
lipofectamine
transfection
