Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line
Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line
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颗粒型日本脑炎病毒包膜蛋白在稳定转染的果蝇细胞系中的表达
作者：
Fuquan Zhang;Wen;Li Zhang;M. Aasa;Hongyi Zhang
研究背景日本脑炎病毒（Japanese encephalitis virus，JEV）是黄病毒科（Flaviviridae）的一种重要蚊媒病原体。它的包膜糖蛋白（E）是决定其致病性和宿主免疫应答的主要因素。结果将JEV E蛋白前膜信号序列和E蛋白编码序列克隆到果蝇表达载体pAc5.1/V5-His中，构建了JEV E蛋白原核表达载体pAc5.1/V5-His。果蝇细胞系S2与该构建体以及提供潮霉素B抗性的质粒共转染。通过免疫荧光、共聚焦显微镜和Western印迹分析，使用三种不同的针对JEV E蛋白的单克隆抗体选择表达JEV E蛋白的细胞系。在潮霉素B存在下，该细胞系在体外维持两个月期间JEV E蛋白产量稳定。结果表明，重组E蛋白具有约50千道尔顿的预期分子量，与所有三种单克隆抗体免疫反应，并发现在细胞质和培养上清。蔗糖梯度离心分析显示分泌的E蛋白产物为颗粒状。它迁移到密度为1.13 g/ml的蔗糖级分。用含有E蛋白颗粒的蔗糖部分免疫的Balb/c小鼠产生特异性抗体。这些数据表明，功能JEV E蛋白表达在稳定的S2细胞株。结论果蝇表达系统是一个更方便，更便宜，更安全的方法来生产的候选疫苗和诊断试剂的JEV。
BackgroundJapanese encephalitis virus (JEV), a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E) is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system.ResultsThe coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line.ConclusionThe Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.
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