登革病毒2型E蛋白基因真核表达和DNA免疫的研究

将编码登革病毒2型(DV2)氨基末端80％的E蛋白的DNA片段克隆到真核表达载体pCXN2 AG强启动子下游,构建成DV2E重组真核表达质粒pCXN-E。间接免疫荧光显示其可在COS-7细胞中表达。ELISA法检测pCXN2-E DNA免疫BALB/c鼠血清中的E抗体变化和维持规律,结果显示三次免疫后2周已有抗体产生,15周时仍维持较高的水平;血清空斑减数中和实验显示其中和滴度高于1:640;流式细胞计数仪(FACS)检测DNA免疫鼠CD4~+、CD8~+T淋巴细胞变化情况,与注射空载体pCXN2的阴性鼠相比,CD4~+淋巴细胞水平略有上升。CD8~+细胞水平有较大升高(p<0.01);动物保护性实验结果显示,当用致死剂量登革病毒攻击免疫鼠时,其保护率为60％。以上结果表明:pCXN2-E在实验动物内表达出的DV2E蛋白可以诱导免疫动物的体液免疫和细胞免疫应答,尤其是MHC-I限制性杀伤性CD8~+T淋巴细胞水平的提高对清除病毒是十分有利的。因此,DV2 E DNA免疫为登革病毒DNA疫苗的发展进行了有益的探索。

Eukaryotic Expressing and the DNA Immunization of E Protein Gene of Dengue virus 2

The gene encoding a strategically truncated E glycoprotein, approximately 80% of the N-terminal sequence was cloned into the eukaryotic expressing plasmid pCXN2 to get the recombinant plasmid pCXN2-E. Indirect immune fluorescence assay showed that the recombinant plasmid pCXN2-E can be expressed in COS-7 cells. The specific antibody against DV2 E was detected by ELISA at 2 weeks after the last inoculation, and maintained to 15 weeks; Plague reduction neutralization test (PRNT) was performed on sera obtained at 2 weeks post inoculation. The sera had high PRNT50 titer which was overl: 640; The percentage of CD4~+ T-lymphocyte subtype cells in immune mice increased compared with the control group. The percentage of CD8~+ T-lymphocyte subtype cells in the group of pCXN2-E was significantly higher than that of pCXN2 group (p<0.01); Mice protection against challenge showed that 60% mice survived. In conclusion: pCXN2-E can induce humoral and celluar immune response. Especially, the raise of CD8~+ CTL is important to clear virus.