传染性胃肠炎病毒(TGEV)双启动子真核表达载体pVAXD-N／S的构建与鉴定  

Construction and Identification of a Dural-promoter Eukaryotic Vector pVAXD-N/S of Transmissible gastroenteritis virus (TGEV)

作　　者：黄小波[1] 邓飞[1,2] 曹三杰[1] 文心田[1] 杨恒[1,3] 李春松[1] 张鑫淼[1] 张亮[1] 

机构地区：[1]四川农业大学动物医学院、动物传染病与基因芯片实验室/动物疫病与人类健康四川省重点实验室,雅安625014 [2]四川省动物疫病预防控制中心 [3]昆明理工大学生命科学与技术学院,昆明650224

出　　处：《农业生物技术学报》2012年第10期1207-1214,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30901084);教育部高等学校校博士点基金项目(No.20095103120006);教育部《长江学者和创新团队发展计划》创新团队项目(No.IRTO848)

摘　　要：猪传染性胃肠炎(TGE)是由猪传染性胃肠炎病毒(TGEV)引起的高度接触性传染病。TGEV的S、N基因具有重要的免疫学功能,构建S、N双基因疫苗将能提供更好的免疫效果。本研究用RT-PCR扩增S基因抗原区(2.1kb,含A、B、C和D完整的抗原位点)和N基因编码区(1.2kb),分别定向插入双启动子真核表达载体pVAXD的多克隆位点(MCS),构建了单价质粒pVAXD-N和pVAXD-S,然后将N基因插入pVAXD-S中,构建了双启动子真核表达质粒pVAXD-N/S。将pVAXD-N/S与对照组(pVAXD-N、pVAXD-S和pVAXD)转染COS-7细胞进行表达鉴定,用RT-PCR可从pVAXD-N/S转染细胞中扩增出S、N两个目的基因,IFA鉴定结果显示,pVAXD-N/S可同时表达S、N两种目的蛋白。初步的小鼠(Mus musculus)免疫试验结果显示,pVAXD-N/S免疫小鼠后第14天即可检测到抗TGEV的IgG抗体,第35天抗体达最高峰,pVAXD-N/S诱导的抗体水平显著高于单基因质粒组pVAXD-N和pVAXD-S的抗体水平(P>0.5),与混合质粒(pVAXD-N+pVAXD-S)诱导的抗体水平相当。本研究结果表明,构建的双启动子表达载体pVAXD-N/S具有S、N基因的双重免疫功能,为TGEV新型双基因疫苗研究提供了基础材料。Porcine transmissible gastroenteritis （TGE） is one important causative enteric infection caused by Transmissible gastroenteritis virus（TGEV）. The S and N genes of TGEV have important immunological function respectively, a DNA vaccine co-expressing S and N gene will provide better immune efficacy for controlling. In this research, the S gene fragment （2.1 kb, including A, B, C and D antigenic sites of the S protein） and the full-length N gene（1.2 kb） of TGEV were amplified by RT-PCR, respectively. The N and S genes were respectively inserted into the multiple cloning sites （MCS） of the dural-promoter eukaryotic vector pVAXD to construct the recombinant plasmid pVAXD-N and pVAXD-S firstly. The N gene was subsequently inserted into the recombinant plasmid pVAXD-S to construct the dural-promoter eukaryotic plasmid pVAXD-N/S. The plasmid pVAXD-N/S and the control plasmids（pVAX-S, pVAX-N and pVAXD） were transfected into COS-7 cells to express. The transcription of the S and N genes could be detected by RT-PCR from transfected COS-7 cells. The expression of pVAXD-N/S in COS-7 cells could react with the rabbit anti-S protein serum、the rabbit anti-N protein serum and the rabbit anti-TGEV serum, respectively, by indirect immunofluorescence assay（IFA）, the fluorescence intensity detected from the pVAXD-N/S transfected COS-7 cells was consistent with that detected from the pVAXD-N group and pVAXD-S group. The results indicated that the dural-promoter eukaryotic vector pVAXD-N/S could express the S protein and N protein at the same time. Six-weeks-old BALB/c mice（Mus musculus） were immunized with the plasmid pVAXD-N/S and the control groups（pVAXD-N, pVAXD-S and pVAXD）. The serum IgG antibody was measured by indirect ELISA. The result showed that specific anti-TGEV serum IgG antibody could be detected at day 14 post-immunization, and the antibody peak was detected at day 35 post-immunization. The IgG antibody level induced by pVAXD-N/S was significantly higher than that induced by the pVAX

关 键 词：猪传染性胃肠炎病毒(TGEV) S基因 N基因 双启动子真核表达载体 

分 类 号：S852.659.6[农业科学—基础兽医学]