含CpG序列PRRSV SD2 E基因真核表达载体质粒构建及表达  

Construction and Expression of Eukaryotic Expression Vector Containing PRRSV SD2 E Gene and CpG Motifs

作　　者：温建新[1] 李俊[2] 周顺[3] 任慧英[3] 刘文华[3] 邹玲[3] 徐文[3] 牛钟相[1] 王金宝[2] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018 [2]山东省农业科学院,山东济南250000 [3]青岛农业大学动物科技学院,山东青岛266109

出　　处：《西北农业学报》2008年第3期1-6,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：山东省自然科学基金项目资助(编号:Z2003D01)

摘　　要：为了提高猪生殖与呼吸综合征病毒山东株(PRRSV SD2株)基因免疫的效果,将PRRSV SD2 E基因插入哺乳动物表达载体PVAX1中,构建出PVAX1-E质粒。再将已筛选出的CpG-ODN序列通过在其两端的粘性酶切末端定向插入含PVAX 1-E的真核表达载体,构建含CpG基因序列真核重组表达质粒CpG-pVAX 1-E。将重组质粒转染COS-7细胞,经RT-PCR检测E基因mRNA的转录和间接免疫荧光试验(IFA)证实:CpG-pVAX 1-E可表达PRRSV GP5蛋白。试验结果为进一步研究PRRSV SD2 ORF5动物免疫反应奠定基础。To improve the effect of the gene immunization against porcine reproductive and respiratory syndrome virus（PRRSV）SD2 we constructed the eukaryotic expression vector CpG-pVAX1-E containing E gene coding region and CpG motif by cloning E gene segment with CpG motif into eukaryotic expression pVAX1 vector. After conformed by identified by enzyme analysis and nucleotide sequencing test , the recombinant expression vector plasmid CpG-pVAX1-E was transfected into COS-7 ：cells by using lipofectamine methods. The ORF5 mRNA of transfected cells were detected by RT-PCR. The transient expression of PRRSV SD20RF5 nucleocapsid protein was detected by indirect immunofluorescence assay（IFA）. In some transfected COS-7 cells, the green fluorescence was showed, thus we can conclude that the eukaryotic expression vector CpG-pVAX1-E was successfully constructed and expressed in vitro, which will lay a foundation for studying on further immunogenicity of PRRSV SD2 ORF5 DNA vaccine.

关 键 词：猪生殖与呼吸综合征病毒山东SD2株 E基因 真核表达载体 瞬时表达 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]