东方马脑炎病毒VLPs表达与免疫原性研究  

Expression and immunogenicity study of virus like particles of eastern equine encephalitis virus

作　　者：马金柱[1,2,3] 王化磊[1] 郑学星[1] 吴红霞[1] 高玉伟[1] 张志芳[4] 杨松涛[1] 夏咸柱[1,3] 

机构地区：[1]军事医学科学院军事兽医研究所,长春130122 [2]黑龙江八一农垦大学生命科学技术学院,大庆163319 [3]东北农业大学动物医学学院,哈尔滨150030 [4]中国农业科学院生物技术研究所,北京100081

出　　处：《免疫学杂志》2014年第6期488-492,共5页Immunological Journal

基　　金：国家科技计划项目(2013BAD12B04)

摘　　要：目的表达东方马脑炎病毒(EEEV)病毒样粒子(VLPs),研究其免疫原性。方法采用PCR方法扩增目的基因,酶切之后连接到供体质粒pFastBacTM1上,构建成重组质粒pFastBacTM1-C-E,然后重组质粒经过基因重组作用与家蚕核型多角体病毒(BmNPV)形成重组病毒BmNPV-C-E,BmNPV-C-E被转染到BmN细胞后以表达VLPs。VLPs鉴定表达正确后,用纯化VLPs与弗氏佐剂乳化成免疫原免疫小鼠,免疫剂量为15μg/只,免疫途径为后肢肌肉注射,免疫2次且间隔时间为14 d。2免后第7天检测免疫小鼠外周血中CD4+T细胞/CD8+T细胞比例和细胞因子IL-2、IFN-γ和TNF-α含量,2免后第14天采用ELISA方法检测免疫小鼠血清内抗体IgG效价。结果研究结果显示实验组小鼠的CD4+T细胞/CD8+T细胞比例和细胞因子IL-2、IFN-γ和TNF-α质量浓度与对照组相比差异均显著(P<0.01),免疫原实验组和VLPs免疫组2免后的血清效价分别为1∶128与1∶64,而对照组小鼠血清中没有产生IgG抗体。结论结果表明BmNPV-C-E能在BmN细胞中成功表达EEEV VLPs,并且VLPs能够刺激小鼠产生明显免疫反应,具有较强免疫原性,这为EEEV新型疫苗研究提供了重要参考。This study designed to explore the immunogenicity of virus like particles （VLPs） of eastern equine encephalitis virus （EEEV）. All encoding protein genes, C-E3-E2-6k-El of EEEV, were amplified using PCR technique. The PCR products were digested and inserted into pFastBac^TM1 plasmid, designated as pFastBae^TM1-C-E. After identification, the pFastBac^TM1-C-E plasmid was recombinated with BmNPV （Bombyx mori nuclear polyhedrosis virus） by genetic recombining method to construct BmNPV-C-E, which was responsible for the production of the recombinant baculovirus. EEEV VLPs were expressed by BmNPV-C-Es transfected BraN cells. After VLPs were detected using the electron microscopy and indirect immunofluorescence （IIF） assay, they were purified by discontinuous sucrose gradient method. The Balb/e mouse was intramuscularly inoculated twice with a dose of 15 μg VLPs plus freund＇s adjuvant at an interval of 14 days in the right and left quadriceps. Seven days after the boost immunization, the ratio of CD4＋ T cell/CD8＋ T cell and the concentrations of IL-2, IFN-γ and TNF- α in peripheral blood of mice were detected; fourteen days after the boost immunization, the titers of IgG antibody were evaluated using ELISA assay. The data showed the ratio of CD4＋ T eell/CD8＋ T cell and the concentrations of IL-2, IFN-γ and TNF-α of experimental group were significant difference compared with the control groups in thewhole experiment （P 〈 0.01）, and the titers of IgG antibody of experimental group and VLP group were 1： 128 and 1：64, respectively, however, no IgG antibody was detected in samples from the control groups. Therefore, EEEV VLPs generated by the baculovirus expression vector system （BEVS） exhibit strong immunogenicity in mice, which could provide a novel,safe, non-replicating and effective vaccine candidate against EEEV infections.

关 键 词：东方马脑炎病毒 载体构建 病毒样粒子 免疫原性 

分 类 号：S852.65[农业科学—基础兽医学]