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Location of Protective Epitopes on the E Protein of Japanese Encephalitis Virus and its Expression

乙型脑炎病毒E蛋白保护性表位的定位及其表达

基本信息

批准号：
61570233
负责人：
YASUI Kotaro
金额：
$ 1.28万
依托单位：
Tokyo Metropolitan Institute for Neurosciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

cDNA of the genomic RNA of Japanese encephalitis(JE) virus were molecularly cloned and sequenced. Amino acid sequences of the E protein were deduced from the base sequnce data.The protein was composed 3 domain structures and epitopes were located mainly on these domains.The E proteins of flaviviruses might have the similar domain structure because amino acid sequence homology of the E proteins was very high between JE virus and other flaviviruses and all cystein residues on the E proteins were completly conserved among the flaviviruses sequenced.The protective epitopes were located restricted sites on the folded E protein.Monoclonal antibody escaped mutants of the protective epitopes were isolated and their base sequences of the E protein gene were analysed.Protective epitopes might be located on the N terminal domain of the E protein because the amino acid replacement of these monoclonal antibody escaped mutants were observed on the N terminal domain.The E proteins from the constructs which contained the E protein gene and other parts of the structural and nonstructural protein genes were expressed by using recombinant baculovirus,SV40 plasmid and in vitro translation system.Among them similar structural E protein to the JE virion , that is natively folded E protein,was expressed from the construct which contained pre M,E and SSl sequences,however unfolded denature form E proteins were expressed from the constructs which containd only E sequence.To express natively folded E protein preM sequence might be important at upstream of the E protein sequence.It became possible to develop the compornent E protein vaccine and diagnostic reagents using the recombinant baculovirus because the animals immunized with the E protein which was expressed by the recombinant baculovirus contained JE virus preM,E and NSl protein genes could produced high titred neutralizing antibodies.

对日本脑炎病毒（JE）基因组RNA的cDNA进行了克隆和序列测定。根据E蛋白的氨基酸序列推断，该蛋白由3个结构域组成，表位主要位于这些结构域上。由于E蛋白的氨基酸序列与其他黄病毒的同源性很高，且E蛋白上的半胱氨酸残基在黄病毒中完全保守，因此黄病毒E蛋白可能具有相似的结构域对E蛋白的N端结构域进行了氨基酸替换，从而确定了E蛋白的N端结构域为保护性抗原表位。E蛋白的N端结构域为E蛋白的N端结构域用重组杆状病毒、SV40质粒和体外翻译系统表达了E蛋白基因及其它部分结构蛋白和非结构蛋白基因，其中与乙脑病毒粒子结构相似的E蛋白，即天然折叠的E蛋白，由含有前M、E和SS1序列的构建体表达，而仅含E蛋白序列的构建体表达了未折叠的E蛋白，表达天然折叠的E蛋白前M序列可能是E蛋白序列上游的重要序列，利用该序列可开发E蛋白疫苗和诊断试剂由于重组杆状病毒表达的E蛋白免疫动物可产生高滴度的中和抗体，因此，重组杆状病毒具有良好的免疫原性。