登革病毒2型NS1蛋白DNA疫苗的构建及其免疫效果观察

Construction and immunization of DNA vaccine containing DV2 NS1 gene

目的 以登革病毒 2型 (denguevirus2 ,DV2 )非结构蛋白 (non structrulprotein 1,NS1)为靶基因 ,构建DV2 NS1的候选DNA疫苗 ;并探讨其在小鼠体内诱导特异性体液免疫和细胞免疫的作用。方法 将登革病毒 2型NS1 NS2a基因片段克隆至含AG强启动子的真核表达载体pCXN2上 ,构建成重组体pCXN2 NS1 NS2a。在体外将重组质粒转染Cos 7细胞 ,间接免疫荧光检测其在真核细胞中的表达。大量提取空质粒和重组质粒 ,进行动物免疫实验。结果 重组质粒可在真核细胞中有效地表达NS1蛋白。免疫接种小鼠后可诱发机体产生针对NS1蛋白的特异性体液免疫和细胞免疫。末次免疫前已有抗体产生 ,4周后达高峰。抗体依赖补体介导的溶细胞作用 (antibody dependentcomple ment mediatedcytolysis,ADCC)试验结果显示产生的抗体在体外具有特异的杀细胞作用。淋巴细胞增殖实验结果显示 ,实验组小鼠的淋巴细胞增殖能力与对照组比较差异有显著性。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 + 、CD8+ 细胞水平有较大升高 (P <0 .0 1)。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 ,有 6 6 .6 %的免疫鼠受到保护。结论 NS1 NS2a基因重组质粒免疫小鼠可以诱?

Objective To develop a DV2-NS1 DNA vaccine, we have constructed a recombinant plasmid encoding NS1 protein gene. Methods DV2 NS1/NS2a gene was cloned into the eukaryotic expressing plasmid pCXN2 to get the recombinant plasmid pCXN2-NS1/NS2a, which was then transfected into Cos-7 cells. A large amount of the plasmids were used to immunize BALB/c mice. Results Indirect immunofluorescence assay showed that the recombinant plasmid pCXN2-NS1/NS2a expressed in Cos-7 cells. The specific antibody against DV2 NS1 was detected by ELISA before the last booster with the recombinant plasmid, and reached its peak at forth week. The NS1 antibody exhibited cytolytic activity against DV2-infected human endothelial cell line derived from umbilical vein(ECV304) in antibody-dependent complement-mediated cytolysis test. The proliferation activity of spleen T lymphocytes in the mice immunized with pCXN2-NS1/NS2a was higher than that in the pCXN2 group. The percentage of CD4 + and CD8 + T-lymphocyte subtype cells in the group of pCXN2-NS1/NS2a was significantly higher than that of pCXN2 group( P <0.01). Mouse protection against challenge showed protection of 66.6%. Conclusion pCXN2-NS1/NS2a can induce both humoral and cellular immune response. The present work may be useful to the development of Dengue virus DNA vaccine.