荧光报告质粒构建及其在布鲁菌细胞侵袭试验中的应用  

Construction of a new GFP plasmid and its primary application in Brucella cell infection

作　　者：钟志军[1,2] 乔凤[2,3] 于爽[1,2] 任志华[1] 徐杰[2] 王玉飞[2] 黄祥明 陈泽良[2] 兰景超 吴孔菊 彭广能[1] 

机构地区：[1]四川农业大学动物医学院,动物疫病与人类健康四川省重点实验室,四川雅安625014 [2]军事医学科学院疾病预防控制所,北京100071 [3]海南省皮肤病医院海南省皮肤性病防治中心,海口570206 [4]成都大熊猫繁育研究基地四川省濒危野生动物保护生物学重点实验室-省部共建国家重点实验室培育基地,成都610081

出　　处：《中国兽医学报》2014年第1期61-65,共5页Chinese Journal of Veterinary Science

基　　金：国家“973“课题(2009CB522602);国家自然科学基金资助项目(31000548);教育部“长江学者和创新团队发展规划”创新团队项目(IRT0848);四川农业大学“双支计划”项目(00270713);成都大熊猫繁育研究基金会项目(CPF研2012-6);四川省教育厅重点资助项目(13ZA0263)

摘　　要：采取质粒shuffling方法,将含GFPmut1的pPS858和能在布鲁菌中复制的质粒pBBR1MCS-2经KpnⅠ酶切后连接,转化DH5α,利用双抗性筛选重组质粒pBBR1-GFP。将荧光质粒转入布鲁菌,获得荧光标记布鲁菌,并用构建好的荧光标记布鲁菌侵染巨噬细胞J774A.1,探讨其用于细胞侵袭试验的可行性。结果显示,成功构建布鲁菌荧光报告质粒pBBR1-GFP,用构建好的荧光标记布鲁菌侵染巨噬细胞后,在细胞内观察到带荧光的布鲁菌,且荧光强度与胞内细菌数成正比。细胞侵袭动态试验结果表明,细菌与细胞共孵育45min后,胞内细菌达到饱和。结果表明,成功构建了布鲁菌的荧光质粒报告系统,可用于布鲁菌细胞侵袭试验。To construct a new plasmid that could replicate in Brucella for pathogenesis study. By using plasmid shuffling method, pPS858 containing GFPmutl and pBBR1MCS2 were digested with Kpn I ; Then the DNA was ligated and transformed into DH5a. The recombinant plasmid pBBR1-GFP was selected by its resistance to kanamycin and gentamycin. Then pBBR1-GFP was transformed into Brucella to test the feasibility of application in invasion assays. A new plasmid pBBR1-GFP was successfully constructed. Brucella with the plasmid could be detected during cell invasion,and furthermore, the intensity of fluorescence is proportional to the number of intracel-lular Brucella. Dynamic cell invasion assays showed that, the bacterial number of intracellular peaks at 45 min post-infection. A new GFP plasmid that could replicate in Brucella was constructed and this plasmid could be used in cell invasion assays.

关 键 词：布鲁菌 荧光报告质粒 侵袭试验 

分 类 号：S852.61[农业科学—基础兽医学]