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

对日本脑炎（乙脑）病毒基因组RNA cDNA进行了分子克隆和测序。根据碱基序列数据推导出E蛋白的氨基酸序列。该蛋白由3个结构域组成，表位主要位于3个结构域上。乙脑病毒与其他黄病毒的E蛋白氨基酸序列同源性很高，且E蛋白上的所有半胱氨酸残基在被测序的黄病毒中完全保守，可能具有相似的结构域结构。保护性表位位于折叠的E蛋白上的限制性位点。分离了E蛋白保护性表位的单克隆抗体逃逸突变体，并分析了其碱基序列。保护性表位可能位于E蛋白的N端结构域，因为这些单克隆抗体逃逸突变体在N端结构域上观察到氨基酸的替换。利用重组杆状病毒、SV40质粒和体外翻译系统，表达了含有E蛋白基因和其他部分结构蛋白和非结构蛋白基因的E蛋白。其中与乙脑病毒粒子结构相似的E蛋白是天然折叠的E蛋白，由含有pre M、E和SSl序列的构建体表达，而未折叠的变性E蛋白则由只含有E序列的构建体表达。在E蛋白序列的上游表达天然折叠的E蛋白preM序列可能是重要的。重组杆状病毒表达的含有乙脑病毒preM、E和NSl蛋白基因的E蛋白免疫动物后，可产生高滴度的中和抗体，从而使利用重组杆状病毒研制E蛋白组分疫苗和诊断试剂成为可能。

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.