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

对乙型脑炎病毒基因组RNA进行了分子克隆和序列测定。该蛋白由3个结构域组成，表位主要位于3个结构域。由于乙脑病毒与其他黄病毒E蛋白的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黄病毒序列中都是完全保守的，所以黄病毒的E蛋白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。我们分离到保护性表位的单抗逃逸突变株，并对其碱基序列进行了分析。保护性表位可能位于N端通过重组杆状病毒表达来自含有E蛋白基因的构建体的E蛋白以及结构蛋白和非结构蛋白基因的其他部分，其中，含有前M、E和SS1序列的重组杆状病毒表达的E蛋白是天然折叠的E蛋白，而只含有E序列的载体表达的是未折叠的变性形式的E蛋白。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。用含有乙脑病毒Prem的重组杆状病毒表达的E蛋白免疫动物，可以产生高滴度的中和抗体。

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.