摘要
将构建的重组质粒pEGFP-N1-P49和pEGFP-N1-P53同时进行双酶切,回收P53基因片段和pEGFP-N1-P49载体,再将P53基因片段重组到pEGFP-N1-P49中,构建旋毛虫ES抗原P53和P49融合基因的真核表达载体pEGFP-N1-P49-P53,利用脂质体法将其转染哺乳动物细胞COS-7。于转染后第24小时在荧光显微镜下观察到发绿色荧光的转基因细胞;Western-blot分析证实目的基因的表达产物具有抗原性。结果表明,成功构建了含有旋毛虫ES抗原P53和P49融合基因的真核表达载体pEGFP-N1-P49-P53,并在COS-7细胞中表达了目的蛋白,并且表达的蛋白可被感染旋毛虫小鼠阳性血清特异地识别。
The constructed recombinant plasmids pEGFP-N1-P49and pEGFP-N1-P53were digested with KpnⅠand BamHⅠ,and P53 gene was inserted into linerized pEGFP-N1-P49 to construct eukaryotic expression vector pEGFP-N1-P49-P53,and then it was transfected into COS-7 cells by means of lipofectamine-mediated method.The transgenic cells displayed bright fluorescence at hour 24 after transfection under fluorescence microscope.Western-blot analysis showed that the expressed protein could react with the sera from mice infected by Trichinella spiralis.It was concluded that the co-expression vector pEGFP- N1-P49-P53 with the P49-P53 fusion gene was constructed and the fusion protein P49-P53 with immunogenicity was expressed in COS-7 cells.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第10期1044-1047,共4页
Chinese Veterinary Science
基金
黑龙江省普通高等学校青年学术骨干支持计划项目(1153G007)
国家科技支撑计划重大项目(2010BAD04B01)
关键词
旋毛虫
P49基因
P53基因
真核表达
Trichinella spiralis
P49 gene
P53 gene
eukaryotic expression
