狂犬病毒融合糖蛋白DNA疫苗及免疫效果的研究

目的：构建含两种狂犬病毒疫苗株的融合糖蛋白基因DNA疫苗并对小鼠的免疫效果进行评价.方法：用RT-PCR法分别扩增和分离出SRV9株和CTN株的狂犬病毒糖蛋白部分表位的基因（SRV9毒株的1～252氨基酸和CTN毒株253～524氨基酸）,构建含融合G蛋白基因的DNA疫苗质粒VR1055-SRV9CTN.碱裂解法大量提取重组质粒并经Sepharose 4FF柱层析纯化后直接肌肉注射免疫NIH小鼠,用ELISA法和淋巴细胞转化实验分别检测质粒DNA疫苗诱导小鼠产生抗狂犬病毒的体液免疫和细胞免疫效果.CTLL-2依赖细胞株/MTT比色法进行IL-2活性测定.结果：VR1055-SRV9CTN DNA疫苗具有较好的诱生狂犬病毒抗体能力.诱导细胞免疫方面,质粒VR1055-SRV9CTN DNA疫苗和空载体对照组的ConA刺激指数存在显著性差异（t=7.201,P=0.000）.小鼠血清中的IL-2活性测定表明DNA疫苗组显著高于空载体组（t=21.616,P=0.000）.CVS株RV脑内攻击保护实验中,疫苗组和对照组小鼠存活率分别为63%、25%,二者间差异具有显著性（t=7.065,P=0.008）.结论：含狂犬病毒糖蛋白基因VR1055-SRV9CTN DNA疫苗既能诱导体液免疫又能诱导细胞免疫,具有较好的免疫保护效果.

Construction of rabies fusion glycoprotein DNA vaccine and its immunological effect

Objective:To construct rabies fusion glycoprotein DNA vaccine and evaluate its immunogenicity in NIH mice.Methods:Part of G of the SRV9 strain(aa 1-252) and the CTN strain(aa 253-504) were cloned and isolated by RT-PCR and a fusion G gene was constructed.The fusion gene was into inserted the eukaryotic expression vector VR1055.Sequence analysis were taken before and after the construction.Large quantities of plasmid was extracted by alkali lysis and purified by Sepharose 4FF chromatography.The plasmids prepared were injected intramuscularly into NIH mice.The induced humoral immune response and cellular immune response were evaluated by ELISA and spleen lymphocyte cells transformation experiment,respectively.IL-2 activity was determined by CTLL-2 cell line/MTT method.Results:The titers of rabies antibodies were detected in the sera of immunized mice.The mean value of ConA stimulated indexes between VR1055(control group) and VR1055-SRV9CTN immunized groups were different(t=7.201,P=0.000).The activities of IL-2 in immunized groups were significantly higher than the control groups(t=21.616,P=0.000).VR1055-SRV9CTN protected 63% of NIH mice against CVS attack,while VR1055 protection rate was 25%.Conclusion:The rabies glycoprotein DNA vaccine could induce homoral and cellular immune responses in NIH mice,showing good protective effect on rabies virus.