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

利用重组DNA技术研制第二代乙脑疫苗。为了获得这一目标的基础信息，分析了乙脑病毒的全核苷酸序列。该病毒基因组由10 976个核苷酸组成，其中5'端非编码区95个碱基，3'端非编码区585个碱基，两个非编码区之间有10 296个碱基形成一个长的开放阅读框架（ORT）。N端氨基酸序列分析表明，乙脑病毒结构蛋白基因存在于ORF的5' 1/4区，序列顺序为C、PreM（M的前体）和E。Sicne E蛋白是一种能产生中和抗体的蛋白质，它在乙脑的保护中起着重要的作用。将E蛋白基因插入到各种重组质粒或病毒中，使E蛋白在转化或感染这些质粒或病毒的细胞中表达。E蛋白分别在重组酵母、哺乳动物细胞、痘苗病毒和家蚕核型多角体病毒中得到表达，但其免疫原性远低于现有疫苗。低免疫原性的原因可能是（1）表达的E蛋白的量，（2）表达的细胞中E蛋白的稳定性，（3）免疫原性对E蛋白的高度构象结构的依赖性，（4）用于免疫的表达的细胞中e蛋白的纯度。这些问题有待于进一步研究，以提高表达水平，纯化表达的E蛋白，并尝试表达E蛋白C端附近的抗蛋白变性的中和表位。

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.