Development of flavivirus vaccine including Japanese encephalitis virus by recombinant DNA technologies

含有病毒包膜蛋白编码序列的重组杆状病毒和牛痘病毒，即。例如，构建了日本脑炎病毒（JEV）前体蛋白、M蛋白和E蛋白的重组质粒。这些在蛋白质的每个编码序列上游具有信号序列的重组体产生生物学和抗原活性的JEV蛋白，其被糖基化并被加工成与真实蛋白相似。我们的结果还表明，重组病毒有效表达E蛋白需要编码E蛋白上游的信号序列，并且当E蛋白的每个表位成功表达时，在重组病毒感染的细胞的表面和培养上清液上检测到该蛋白。重组病毒感染细胞后释放的E蛋白为小颗粒，与M蛋白呈寡聚体或异源寡聚体，感染动物或接受重组病毒产物的动物对这些E蛋白的免疫应答相似。 关于我们 包括显著产生中和抗体作为JEV感染和攻击测试的鱼藤蛋白成功地通过使用小鼠进行。E蛋白寡聚体和与M蛋白组成的异源寡聚体颗粒是比单体E更好的免疫原，并可诱导产生中和和血凝抑制等生物活性抗体。通过对JEV单克隆逃逸突变株和毒株的分析，确定了E蛋白上JEV特异性中和表位和毒株特异性表位的位置。成功地制备了与部分JEV E蛋白（仅含特异性抗原表位）融合的颗粒状HBsAg，为进一步研究制备颗粒状E蛋白和仅含特异性抗原表位的HBsAg颗粒以有效诱导中和抗体的方法奠定了基础这些结果为利用重组病毒技术研制JEV及其他黄病毒的新疫苗提供了基础信息。少

Recombinant baculo- and vaccinia viruses containing the coding sequences of the viral envelope proteins, i. e., preill, M, and E protein of Japanese encephalitis virus (JEV) were constructed. These recombinants which had a signal sequence at the upstream of each coding sequence of the proteins produced biologically and antigenica-lly active JEV proteins which were glycosylated and processed as similar to the authentic proteins. Our results also indicate that efficient expression of the E protein by recombinant virus requires the signal sequence which is encoded upstream of E protein and when each epitope of E protein is successfully expressed, this protein is detected on surfaces and culture supernatant of recombinant virus infected cells. The E proteins released from recombinant virus infected cells were small particles and oligomers or heterooligomers with M protein.Animals infected with these recombinants or received the product of them showed similar immune response against these p … More roteins including remarkable production of neutralizing antibodies as JEV infection and challenge testing was successfully done by using mice. The E protein oligomer and hetero oligomer particles composed with M protein were more good immunogens and elicited biologically active antibodies such as neutraling and hemaggulutination inhibition than monomer E.Locations of JEV specific and WN subgroup virus specific neutralizing epitopes and strain specific epitopes on the E protein were detenaned by analyzing monoclonal escaped mutants and strains of JEV. HBsAgs fused with a part of JEV E protein containing only specific epitopes were successfully produced in particulate forms.As a conclusion, we could develop the methods which could produce particulate E proteins and also HBsAg particles with only specific epitopes to induce neutralizing antibody effectively. From these results we could show the basic informations to develop new vaccines for JEV and other flaviviruses by recombinant virus technologies. Less