欧洲型PRRSV重组腺病毒疫苗的构建及免疫效果评价  

Construction of a recombinant adenovirus vaccine against European PRRSV and assessment of its immunogenicity

作　　者：曹亮[1,2] 孙文超[2,3] 解长占 陈兴[1,2] 韩继成[2] 郭海宁[1,2] 肖朋朋[2] 张萍[1,2] 马海彬[2] 南福龙 崔卓栋 鲁会军[2] 钱爱东[1] 金宁一[2] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]广西大学动物科学技术学院广西南宁530004

出　　处：《中国病原生物学杂志》2015年第11期961-965,975,共6页Journal of Pathogen Biology

基　　金：国家863计划项目(No.2011AA10A208);吉林省科技厅项目(No.20140309024NY)

摘　　要：目的构建欧洲型PRRSV重组腺病毒疫苗并对其免疫原性进行分析,为欧洲型PRRSV的感染预防提供依据。方法 PCR扩增欧洲型蓝耳病病毒ORF3、ORF5目的基因,将其连接到腺病毒穿梭载体,构建重组腺病毒穿梭质粒pacad-EU-ORF3、pacad-EU-ORF5、pacad-EU-ORF3-ORF5。将重组腺病毒穿梭质粒与骨架质粒共同转染至HEK 293细胞,构建重组腺病毒,并对构建的病毒用Western blot方法进行验证。用构建的重组腺病毒免疫BALB/c小鼠,通过淋巴细胞增殖试验对重组疫苗免疫效果进行初步评价。结果 Western blot显示重组腺病毒可稳定表达目的蛋白ORF3和ORF5,分子质量单位分别为30ku和22ku,与预期值相符。免疫小鼠淋巴细胞增殖试验显示重组腺病毒刺激指数均为PBS组1.5倍以上(P<0.05),且rAd-EU-ORF3-ORF5组显著高于rAd-EU-ORF3组和rAd-EU-ORF5组,表明重组腺病毒疫苗能够诱导机体淋巴细胞增殖,增强机体的细胞免疫应答水平。结论成功包装出重组腺病毒rAdEU-ORF3、rAd-EU-ORF5和rAd-EU-ORF3-ORF5,动物实验显示其具有良好的免疫原性。Objectives To construct a recombinant adenovirus vaccine against the European porcine reproductive and re- spiratory syndrome virus （European PRRSV） and to analyze its immunogenicity in order to provide evidence for the pre- vention of European PRRSV infection. Methods The ORF3 gene and the ORF5 gene of the European PRRSV were amplified with RT-PCR and ligated to a shuttle vector, and the recombinant adenovirus plasmids pacad-EU-ORF3, pacad- EU-ORF5, and pacad-EU-ORF3-ORF5 were constructed. The constructed plasmids were co-transfected into HEK293 cells with the plasmid pacad5 9.2--100 to obtain a recombinant adenovirus by homologous recombination. The target pro- teins expressed by the recombinant adenovirus were verified with Western blotting. BALB/c mice were immunized with the recombinant adenovirus and the immunogenicity of the vaccine was evaluated based on lymphocyte proliferation. Re- sults The GP3 and GP5 proteins were stably expressed according to Western blotting. A lymphocyte proliferation test indicated that mice receiving the recombinant adenoviruses had a stimulation index of 2. 425, 2. 230, and 3. 477. Mice re- ceiving the recombinant adenoviruses had a stimulation index 1.5 times that of mice receiving PBS （P〈0.05）, and mice immunized with rAd-EU-ORF3-ORF5 had a higher stimulation index than did mice immunized with rAd-EU ORF3 or rAd-EU-ORF5. Results indicated that recombinant adenovirus vaccines were able to induce lymphocyte proliferation and enhance the body＇s immune response. Conclusion The recombinant adenovirus vaccines rAd-EU-ORF3, rAd-EU-ORF5, and rAd-EU-ORF3-ORF5 were successfully constructed. Animal experiments indicated that the recombinant ade novirus vaccines had good immunogenieity.

关 键 词：EU-PRRSV 重组腺病毒 构建 免疫评价 

分 类 号：R373[医药卫生—病原生物学]