登革-日本脑炎嵌合病毒样颗粒的设计与构建  

Design and construction of chimeric virus-like particles of Dengue and Japanese encephalitis virus

作　　者：胡珍[1] 尚伟龙[1] 张俊磊[1] 朱军民[1] 杨杰[1] 刘佳[1] 方昕[1] 袁文常[1] 程航[1] 胡晓梅[1] 饶贤才[1] 

机构地区：[1]第三军医大学微生物学教研室重庆市微生物工程实验室,重庆400038

出　　处：《中国预防医学杂志》2011年第8期645-649,共5页Chinese Preventive Medicine

基　　金：国家自然科学基金(310708111);重庆市自然科学基金(CSTC;2009BB5015);第三军医大学科研基金(2009XYY04;2009XHG03)

摘　　要：目的设计构建登革-日本脑炎嵌合蛋白表达载体,制备嵌合病毒样颗粒。方法 根据登革病毒样颗粒的形成机制及日本脑炎病毒中和性抗原表位的分布,设计并构建登革-日本脑炎嵌合蛋白表达载体,转染BHK-21细胞,筛选能表达目标蛋白的G418抗性克隆,收集细胞培养上清,纯化病毒样颗粒,用Western-blot和电镜检测病毒样颗粒的形成。结果用RT-PCR扩增、酶切和连接等分子生物学方法成功构建了序列及阅读框均正确的重组表达质粒pCI-SMEJ-14#;将该质粒转染BHK-21细胞,经0.6 g/LG418筛选获得4个能表达目标嵌合蛋白的克隆,从培养的细胞上清中能纯化出直径30~100 nm的病毒样颗粒。结论 所设计的登革-日本脑炎病毒嵌合蛋白能在BHK-21细胞中产生病毒样颗粒,为制备新一代登革-日本脑炎嵌合型疫苗奠定了良好基础。Objective To construct the chimeric virus-like particles（VLPs） of Dengue and Japanese encephalitis viruses.Methods An expression vector was constructed by the formation of a Dengue virus（DV） and the gene of neutralizing epitopes of Japanese encephalitis envelope E.The constructed vector was transfected in BHK-21 cells and selected by G418 sulfate.The supernatant of selected cell culture was collected for chimeric VLPs purification.The recombinant VLPs were characterized by western blot and transmission electron microscope（TEM）.Results The recombined expression plasmid of pCI-SMEJ-14# carrying the fusion gene of interest was constructed and confirmed by restriction enzymes analysis and DNA sequencing.With successful BHK-21 cells transfection and 0.6 mg/ml of G418 selection,4 clones secreted the fusion proteins of Dengue and Japanese encephalitis virus as desired.Diameters of 30-100 nm VLPs could be assembled by the expressed proteins under TEM.Conclusion The chimeric protein of VLPs may be valuable in preventing Dengue and Japanese encephalitis virus infection.

关 键 词：登革病毒 日本脑炎病毒 病毒样颗粒 

分 类 号：R392[医药卫生—免疫学]