湖羊myogenin重组基因表达及其亚细胞定位的研究  

Expression and Subcellular Localization of Hu Sheep Myogenin and EGFP Fusion Protein

作　　者：许峰[1] 秦玉蓉[1] 阴彦辉[1] 张振韬[1] 张亚妮[1] 高波[1] 杜立新[2] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009 [2]中国农业科学院畜牧研究所国家畜禽分子遗传育种中心,北京100193

出　　处：《中国农学通报》2011年第7期309-313,共5页Chinese Agricultural Science Bulletin

基　　金：国家转基因重大专项"优质转基因肉羊新品种培育"(2008ZX08008-003;2009ZX08008-003B)

摘　　要：为构建湖羊肌细胞生成素(myogenin,MyoG)与增强型绿色荧光蛋白(EGFP)的融合蛋白真核表达载体,分析EGFP-MyoG融合蛋白在NIH-3T3细胞的表达及亚细胞定位。利用PCR技术扩增出MyoG基因完整的CDS,克隆至真核表达载体pEGFP-C1,转染NIH-3T3细胞,荧光显微镜下观察EGFP-MyoG融合蛋白的亚细胞定位,并通过RT-PCR、western blot检测蛋白的表达。酶切鉴定和DNA序列分析均证实,MyoG基因已正确插入pEGFP-C1中,获得融合蛋白表达载体pEGFP-C1-MyoG,转染NIH-3T3细胞48h后,RT-PCR检测到约680bp的特异性条带,western blot检测出相对分子量约为53kD的融合蛋白,荧光显微镜分析表明,MyoG蛋白定位于细胞核。说明融合蛋白EGFP-MyoG在NIH-3T3细胞中成功表达,并定位于细胞核。To construct the eukaryotic expression vector for enhanced green fluorescent protein （EGFP） fused with Hu Sheep myogenin （MyoG） for investigating the subcellular localization of EGFP-MyoG fusion protein in the transfected NIH-3T3 cells.MyoG gene was amplified by PCRfrom pMD19-T-MyoG and inserted into the eukaryotic expression vector pEGFP-C1 to construct fusion protein expression vector pEGFP-C1-MyoG.Then NIH-3T3 cell was transfected with the constructed recombinant plasmid pEGFP-C1-MyoG.48 hours after transfection,the subcellular localization of the fusion protein was monitored by fluorescence microscopy and the expression of MyoG was detected by RT-PCR and western blot.Enzyme,sequencing analysis indicated that the MyoG gene had been correctly inserted into the pEGFP-C1.RT-PCR detected specific 680 bp band,indicating the expression of fusion protein in RNA level.Western blot analysis confirmed that the EGFP-MyoG fusion protein of Mr 53 kD was detected in transfected NIH-3T3 cells.Fluorescence observed under fluorescence microscopy showed that EGFP-MyoG protein was positioned in the nucleus.The fusion protein EGFP-MyoG was successfully expressed in NIH-3T3 cells,and it was positioned in nucleus in single cell level.

关 键 词：湖羊 MYOG基因 EGFP蛋白 亚细胞定位 

分 类 号：S823.9[农业科学—畜牧学]