Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of economic losses in pigs (Sus domesticus), causing severe reproductive failure in pregnant sows and boars, and respiratory problems in young pigs. This study was aimed to detect the expression of the recombinant co-expressing GP3 and GP5(N-glycosylation protein) and M(membrane protein) fusion protein of PRRSV in PK-15 cells (Porcine kidney cells), and to detect their immunogenicity. The eukaryotic recombinant plasmid pcDNA3. 1-ORF3-ORF5-ORF6 was transfected into PK-15 cells by lipofectamineTM 2000 reagent, the stable expressive cells were selected (72 h) by G418 (μg/mL). We designed three pairs of primers based on the published sequences of PRRSV, the 0RF3 and 0RF5 and ORF6 was transfected into PK-15 cells, and then detected by RT-PCR, the GP3-GP5-M fusion protein was detected by IFA (indirect immunofluorescence assay). The immunogenicity of GP3-GP5-M was detected by Western blot and ELISA (enzyme linked immunosorbent assay). SIP (the ratio of sample/postive control) reached 1.80 after the second immunization, and lasted for at least 1 monthes. The results showed that the fusion protein GP3-GP5 (N- glycosylation protein)-M (membrane protein) could express in mammalian cells. Moreover, the fusion protein showed good immunogenieity. The recombinant plasmid pcDNA3A-ORF3-ORF5-ORF6 may be an attractive candidate vaccine for the prevention and control of PRRS.%猪繁殖与呼吸综合征病毒(PRRSV)是一种严重危害养猪业发展的传染病,为了PRRSV GP3-GP5(N-糖基化蛋白)-M(膜蛋白)融合蛋白在哺乳动物细胞中的表达及其免疫原性,本研究利用脂质体法将构建好的真核重组表达质粒pcDNA3.1-ORF3-ORF5-ORF6转染猪肾细胞(PK-15细胞),经G418(600 μg/mL)筛选(72 h)获得稳定表达PRRSV GP3-GP5-M融合蛋白的稳定细胞株.以RT-PCR分析目的基因转录；间接免疫荧光检测融合蛋白表达；Western blot和小鼠(Mus musculus)免疫试验检测融合蛋白免疫原性.结果表明,RT-PCR检测到3种目的基因的转录；间接免疫荧光检测到融合蛋白得以表达,Western blot检测到融合蛋白可与阳性猪血清发生特异性反应.小鼠免疫试验二免后S/P=样本/阳性对照的比率,达到1.80(S/P＞0.4为阳性),并且至少可以持续1个月.本研究成功实现了PRRSV GP3-GP5-M 融合蛋白在哺乳动物细胞中的表达,证实PRRSV GP3-GP5-M融合蛋白具有良好的免疫原性,为PRRSV 多基因核酸疫苗研制提供了基础资料.
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