新城疫病毒V和W基因在DF-1细胞中的真核表达  

Expression of Recombinant Newcastle Disease Virus V and W Genes in DF-1 Cells

作　　者：汪招雄[1] 康银峰[2] 孙敏华[2] 高诗敏[2] 谢鹏[2] 任涛[2] 

机构地区：[1]长江大学动物科学学院,湖北荆州434025 [2]农业部兽用疫苗重点创制实验室,华南农业大学兽医学院,广东广州510642

出　　处：《长江大学学报（自科版）（中旬）》2013年第8期37-41,7,共5页Journal of Yangtze University(Nature Science Edition)

基　　金：国家自然科学基金项目(3107231);博士点专项基金项目(20124404110016)

摘　　要：采用2轮PCR扩增获得新城疫病毒疫苗株LaSota和强毒株GM V和W基因全长,分别克隆到pMD19-T载体上,然后亚克隆到真核表达载体pEGFP-N1,重组质粒经过酶切,PCR鉴定及测序,筛选出阳性重组质粒,分别命名为pEGFP-La V、pEGFP-La W、pEGFP-GM V和pEGFP-GM W。将各重组质粒分别转染到DF-1细胞,于转染后12、24h和36h在荧光倒置显微镜下观察绿色荧光蛋白GFP在细胞中的表达情况。结果显示,各质粒转染DF-1细胞后在12h就可以见到绿色荧光蛋白,随着时间的延长,绿色荧光蛋白荧光强度增加,到36h后达到最强,而对照组始终没有见到GFP蛋白的表达,表明在转染的阳性细胞中融合蛋白pEGFP-La V、pEGFP-La W、pEGFP-GM V和pEGFP-GM W均能够有效的表达,该结果为新城疫病毒V和W基因的功能研究奠定了基础。The V and W genes of the virulent NDV strain GM and lentogenic strain LaSota were acquired by gene mutation technique of two times polymerase chain reaction(PCR).The four genes were coloned into vector pMD-18 Tand then were sub-coloned into vector pEGFP-N1.The reconstituted plasmid pEGFP-La V,pEGFP-La W,pEGFPGM V and pEGFP-GM W were established by sequences anlysis.Green fluorescent protein(GFP)were expressed when the four plasimds were transfected into DF-1 cells,the intensity of GFP increased as time went on,and the intensity was highest at 36 hafter the plasimds were transfected into DF-1 cells while the control cells did not express any GFP.The results showed that the fusion protein W and V could express after the plasimds pEGFP-La V,pEGFP-La W,pEGFP-GM V and pEGFP-GM W were transfected into DF-1 cells.It laid a good foundation for studying the function of NDV V and W protein.

关 键 词：新城疫病毒 V基因 W基因 鸡胚成纤维细胞 表达 

分 类 号：Q786[生物学—分子生物学]