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

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.