减毒沙门菌递呈的猪传染性胃肠炎病毒S基因与猪IL-6基因双启动子DNA疫苗的构建  

Construction of attenuated SalmonellaTyphimurium harbouring a dual-promoter DNA vaccine coexpressing S gene of transmissible gastroenteritis virus and porcine interleukin 6

作　　者：黄小波[1] 李春松[1] 曹三杰[1] 文心田[1] 张鑫淼[1] 杨恒[1,2] 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,四川雅安625014 [2]昆明理工大学生命科学与技术学院,云南昆明650224

出　　处：《中国兽医科学》2012年第9期921-926,共6页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(30901084);教育部高等学校博士点基金项目(20095103120006);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848)

摘　　要：从猪传染性胃肠炎病毒克隆质粒pMD19T-S中扩增了2.1kb的S基因片段(含完整的A、B、C、D 4个抗原位点序列),以RT-PCR从猪外周血淋巴细胞中扩增了猪白细胞介素6(IL-6)(658bp),插入同一双启动子真核表达载体pVAXD的2个多克隆位点,构建了共表达S与IL-6基因的双启动子表达质粒pVAXD-S-IL-6,转染COS7细胞进行转录和表达鉴定。将质粒pVAXD-S-IL-6电转化减毒鼠伤寒沙门菌SL7207,筛选获得重组菌株SL7207(pVAXD-S-IL-6),并对重组菌株SL7207(pVAXD-S-IL-6)的体外稳定性和口服小鼠的安全性进行了鉴定。结果表明该双启动子真核表达质粒pVAXD-S-IL-6构建成功,RT-PCR检测到转染的COS-7细胞中2个目的基因的转录,间接免疫荧光试验结果显示表达产物可与猪抗猪传染性胃肠炎病毒血清发生特异性反应。重组菌株SL7207(pVAXD-S-IL-6)在含卡那霉素的抗性培养基中稳定性好,以1×109 CFU灌胃接种小鼠具有良好的安全性。本研究构建了猪传染性胃肠炎病毒的新型口服疫苗株SL7207(pVAXD-S-IL-6),为开展其免疫原性评价奠定了基础。A 2.1 kb DNA fragment encoding the S glycoprotein（including A,B,C,and D antigen sites） of transmissible gastroenteritis virus（TGEV） was amplified from the plasmid pMD19T-S,and porcine interleukin 6（IL-6） gene was amplified from porcine peripheral blood lymphocytes by RT-PCR.The two gene fragments were inserted into two multiple cloning sites of the dual-promoter eukaryotic expression vector pVAXD to construct the recombinant plasmid pVAXD-S-IL-6.The transcription and expression of the plasmid pVAXD-S-IL-6 in COS-7 cells was identified.The recombinant plasmid pVAXD-S-IL-6 was transformed into attenuated Salmonella Typhimurium SL7207 competent cells by electroporation to construct recombinant strain SL7207（pVAXD-S-IL-6）.The stability of pVAXD-S-IL-6 in SL7207（pVAXD-S-IL-6）cultivated in vitro was detected. BALB/c mice were inoculated orally with SL7207（pVAXD-S-IL-6） to detect the safety and stability in vivo.The results showed that the recombinant plasmid pVAXD-S-IL-6 was constructed successfully,and the transcription and expression of the two target genes in the COS-7 cells were verified by RT-PCR and indirect immunofluorescence assay.The recombinant bacteria SL7207（pVAXD-S-IL-6） was highly stable cultivated in kanamycin resistance medium in vitro and was safe to mouse at a dosage of 1×10~9CFU.This study provided a novel oral vaccine strain SL7207（pVAXD-S-IL-6） of TGEV,which laid a foundation for evaluating the oral immunogenicity of the vaccine.

关 键 词：猪传染性胃肠炎病毒 S基因 IL-6基因 减毒鼠伤寒沙门菌 构建 鉴定 

分 类 号：S852.612[农业科学—基础兽医学] S852.659.6[农业科学—兽医学]