Development of the second generation Japanese encephalitis vaccine by recombinant DNA technology

应用重组DNA技术研制第二代流行性乙型脑炎疫苗。为了获得这一目标的基本信息，对乙脑病毒的全核苷酸序列进行了分析。病毒基因组由10,976个核苷酸组成，其中95个碱基位于5‘非编码区，585个碱基位于3’非编码区，10,296个碱基在两个非编码区之间形成一个长的开放阅读框架(ORT)。乙脑病毒结构蛋白N端氨基酸序列分析表明，结构蛋白基因位于ORF的5‘1/4位，顺序依次为C、Prem(M的前身)、E蛋白能产生中和抗体，对乙脑病毒的保护作用最大。E蛋白在重组酵母、哺乳动物细胞、痘苗病毒和家蚕核型多角体病毒中均有表达，但其免疫原性明显低于现有疫苗。免疫原性低的原因可能是(1)表达的E蛋白的量，(2)E蛋白在表达细胞中的稳定性，(3)免疫原性依赖于E蛋白的高构象结构，(4)用于免疫的表达细胞中E蛋白的纯度。这些问题需要通过进一步的研究来提高表达水平，纯化表达的E蛋白，并尝试表达出存在于E蛋白C端附近的抗蛋白质变性的中和表位。

Studies were made to develop the second generation Japanese encephalitis (JE) vaccine by recombinant DNA technology. In order to obtain basic information for this objective, complete nucleotide sequence of JE virus was analyzed. The virus genome consisted of 10,976 nucleotides, of which 95 bases existed in 5' noncoding region, 585 bases at 3' noncoding region, and 10,296 bases formed a long open reading frame (ORT) between both noncoding region. The N-terminal amino acid sequences of the structural proteins of JE virus showed that the structural protein gene existed in the 5' 1/4 of the ORF in the order of C, PreM (precursor of M), and E. Sicne E protein is known to produce neutralizing antibodies which play most important roles in the protection of je, e protein gene was inserted into various recombinant plasmids or viruses in order to express the E protein in the cells transformed or infected with these plasmids or viruses. The expression of E protein was detected in recombinant yeast, mammalian cells, vaccinia virus and bombix mori nuclear polyhedrosis virus, respectively, however, the immunogenicity of these expressed.E protein was much lower than that of the current vaccine in terms of eliciting neutralizing antibodies. The reason of the low immunogenicity could be (1) amount of the expressed E protein, (2) stability of the E protein in the expressed cells, (3) dependence of immunogenicity on the highly conformational structure of the E protein, (4) purity of the e protein in the expressed cells used for immunization. These problems should be overcome by further studies to improve the levels of expression, purification of the expressed E protein, and trying to express the neutralizing epitope which is resistant to protein denaturation and exists near C-terminal of the E protein.