Yang Dong-Kun, Kweon Chang-Hee, Kim Byoung-Han, Lim Seong-In, Kwon Jun-Hun, Kim Seong-Hee, Song Jae-Young, Han Hong-Ryul
National Veterinary Research and Quarantine Service, Ministry of Agriculture and Forestry, Anyang, Korea.
J Vet Sci. 2005 Jun;6(2):125-33.
Genes encoding for the premembrane and envelope (prME), envelope (E) and nonstructural protein (NS1) of Japanese encephalitis virus (JEV) were cloned. Each protein was expressed in baculovirus expression system. Of the three proteins expressed in baculovirus system, only prME had hemagglutination activity. The prME (72 and 54 kDa), E (54 kDa) and NS1 (46 kDa) proteins could be detected by Western blotting in the recombinant virus infected cells. Immunogenicity of the recombinant proteins obtained from infected Spodoptera frugiperda (Sf-9) cells was examined in mice. The 3 week-old ICR mice immunized intraperitoneally with three recombinant proteins three times were challenged with a lethal JEV. A survival rate was increased from about 7.7% in unimmunized mice to 92.3% in E+prME and only E groups. The complete protection was shown in prME and live vaccine inoculated groups, respectively. We also measured neutralizing antibody and three immunoglobulin subtypes of IgG1, IgG2a and IgG2b in the sera of mice before and after challenge. Titers of IgG1 antibodies were approximately two to three times higher than that of IgG2b antibodies in all the immunized groups as compared to the control group. However, IgG2a antibody level somewhat increased after challenge, indicating T-helper type 1 (Th1) cell response. The results of this study can provide useful information for developing efficacious subunit vaccine against JEV.
克隆了编码日本脑炎病毒（JEV）前膜和包膜（prME）、包膜（E）和非结构蛋白（NS1）的基因。每种蛋白都在杆状病毒表达系统中表达。在杆状病毒系统中表达的三种蛋白中，只有prME具有血凝活性。通过蛋白质印迹法可在重组病毒感染的细胞中检测到prME（72 kDa和54 kDa）、E（54 kDa）和NS1（46 kDa）蛋白。在小鼠中检测了从感染的草地贪夜蛾（Sf-9）细胞中获得的重组蛋白的免疫原性。用三种重组蛋白对3周龄的ICR小鼠进行三次腹腔免疫，然后用致死剂量的JEV进行攻击。未免疫小鼠的存活率约为7.7%，而E+prME组和仅E组的存活率提高到了92.3%。prME组和活疫苗接种组分别显示出完全保护作用。我们还测量了攻击前后小鼠血清中的中和抗体以及IgG1、IgG2a和IgG2b三种免疫球蛋白亚型。与对照组相比，所有免疫组中IgG1抗体的滴度大约是IgG2b抗体滴度的两到三倍。然而，攻击后IgG2a抗体水平有所升高，表明存在1型辅助性T细胞（Th1）反应。本研究结果可为开发有效的抗JEV亚单位疫苗提供有用信息。
