表达载体pCAGGS显著增强禽流感DNA疫苗的免疫保护效果  

Enhanced Protective Efficacy of Avian Influenza DNA Vaccine with Expressive Vector pCAGGS

作　　者：姜永萍[1] 张洪波[1] 步志高[2] 李呈军[1] 赵有淑[1] 王秀荣[1] 于康震[3] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室,哈尔滨150001 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150001 [3]农业部畜牧兽医总站,北京100026

出　　处：《中国农业科学》2006年第4期825-830,共6页Scientia Agricultura Sinica

基　　金：国家重点基础研究规划"973"项目(G1999011902)资助

摘　　要：目的将A/Goose/GuangDong/1/96(H5N1)[GD/1/96(H5N1)]的HA基因插入鸡β-actin启动子高效真核表达载体pCAGGS,构建了DNA疫苗质粒pCAGGHA5,以提高H5亚型禽流感DNA疫苗的表达水平和免疫保护效果。方法将pCAGGHA5和表达GD/1/96(H5N1)HA基因的质粒pCIHA5通过间接免疫荧光法和Western-blot分析检测转染293T细胞后瞬时表达的HA抗原蛋白,随之将pCAGGHA5及pCIHA5分别以100μg和10μg剂量一次免疫3周龄SPF鸡,4周后用100LD50的HPAIVGD/1/96(H5N1)鼻腔途径进行攻击。结果间接免疫荧光法和Western-blot分析表明2种表达质粒均可正确表达H5亚型HA抗原蛋白,载体pCAGGS表达水平显著高于载体pCI;免疫SPF鸡后,100μgpCAGGHA5可形成5/5的免疫保护,100μgpCIHA5可形成2/4的免疫保护,10μgpCAGGHA5可形成对免疫鸡5/5的免疫保护,而10μgpCIHA5则基本不能形成免疫保护,pCAGGHA5诱导的HI抗体水平远远高于pCIHA5。结论鸡β-actin启动子表达载体pCAGGS可显著提高HA基因体外表达水平和H5亚型禽流感DNA疫苗诱导的保护性抗体免疫反应水平,增强免疫保护效果。[Objective] To improve the protective efficacy of H5 subtype avian influenza DNA vaccine, the HA gene of A/Goose/GuangDong/1/96（H5N1） [GD/1/96（H5N1）] was inserted into pCAGGS, which contains chicken 13 -actin promoter, and was designated as pCAGGHA5, [Method] The monolayer 293T cells were transfected by pCAGGHA5 and pCIHA5, which contained HA gene of GD/1/96（H5N1）. The HA protein expression was detected by indirect immunofluorescence assay 24-48 h after transfection and confirmed by western blotting analysis. To evaluate the vaccine efficacy, groups of 3-week-old SPF chickens were intramuscular inoculated with 100 μg or 10 μg of pCIHA5, pCAGGHA5, respectively. A group of chickens were injected with 200 μl PBS as control. Four-weeks after the single vaccination, all chickens were challenged with 100 LD50 of highly pathogenic GD/1/96（H5N1）, [Results] In vitro expression results indicated the gene in the pCAGGS vector was better expressed than that in the pCI vector. The chickens in the 100μg and 10μg pCAGGHA5 immunized groups were completely protected from virus challenge （no disease signs, no virus shedding and no deaths）, and 100μg pCIHA5 immunized groups only provided partial protection, while the chickens in the pCIHA5 immunized group and the control group died within ten days. [Conclusion] Results shown that expressive vector optimization enhanced the protective efficacy of H5 subtype avian influenza DNA vaccine.

关 键 词：禽流感 H5亚型 鸡β-actin启动子 DNA疫苗 免疫保护 

分 类 号：S852.5[农业科学—基础兽医学]