猪繁殖与呼吸综合征病毒ORF5基因疫苗的构建及其在Marc-145细胞中的表达  

Construction of eukaryotic expression plasmid of PRRSV-ORF5 gene and its expression in Marc-145 cell line

作　　者：希尼尼根[1] 程安春[1] 汪铭书[1] 陈希文[1] 豆文波[1] 李雪梅[1] 刘伍梅[1] 刘菲[1] 张平英[1] 陈孝跃[1] 

机构地区：[1]四川农业大学动物科技学院动物疫病与人类健康四川省重点实验室

出　　处：《中国兽医学报》2008年第3期223-226,共4页Chinese Journal of Veterinary Science

基　　金：教育部“新世纪优秀人才支持计划”资助项目(NCET-04-0906/NCET-06-0818);四川省重大科技攻关资助项目(01NG018-01/05ZQ026-038/2006ZD5-003-01);四川省重点建设学科资助项目(SZD0418)

摘　　要：根据猪繁殖与呼吸综合征病毒(PRRSV)LV和VR-2332株的核苷酸序列,设计合成了1对特异性引物,对PRRSV四川分离株SC1和SC2进行RT-PCR,扩增出病毒ORF5基因。利用真核表达质粒pcDNA3.1(+)成功构建了PRRSV四川分离株ORF5基因疫苗pcDNA-PRRSV-SC1-ORF5和pcDNA-PRRSV-SC2-ORF5。通过脂质体转染法和电穿孔转染法将pcDNA-PRRSV-SC2-ORF5转染Marc-145细胞,间接免疫荧光技术跟踪监测ORF5基因的表达情况,结果表明,脂质体转染法和电穿孔转染法分别在转染26 h和23 h后检测到ORF5基因特异性表达产物,且随着培养时间的延长,特异性表达产物增多,两者均在56 h达到高峰;这证明pcDNA-PRRSV-SC2-ORF5在Marc-145细胞中已高效表达,为PRRSV基因疫苗在临床上应用提供了实验依据。A pair of primers were designed and synthesized according to nucleotide of porcine reproductive and respiratory syndrome virus LV and VR-2332,and DNA fragments of ORF5 gene of PRRSV Sichuan isolates were amplified by RT-PCR.ORF5 of PRRSV Sichuan isolates were inserted into the eukaryotic expression plasmid pcDNA3.1（＋） to construct the recombinant plasmid pcDNA-PRRSV-SC1-ORF5 and pcDNA-PRRSV-SC2-ORF5,then pcDNA-PRRSV-SC2-ORF5 was expressed in the Marc-145 cell line by cytofecteneTM transfection reagent kit processing and electroporation.ORF5 expression was detected by means of immunofluorescence.An intense fluorescence was observed 23 h post transfection in the Marc-145 cells transfected by electroporation,which is 3 h earlier than that processed by cytofecteneTM transfection reagent.The highest expression product was detected 56 h post transfection in the cells transfected with cytofecteneTM transfection reagent or electroporation.Results indicated that PRRSV-SC2-ORF5 gene could be expressed efficiently in the Marc-145 cells,suggesting that the ORF5 of PRRSV should be a good candidate for its gene vaccine.

关 键 词：PRRSV ORF5 基因疫苗 Mare-145细胞 

分 类 号：S852.43[农业科学—基础兽医学] S852.65[农业科学—兽医学]