表达猪流感病毒血凝素基因的重组腺病毒的构建及免疫原性分析  

Construction of Recombinant Adenovirus Expressing Hemagglutinin Gene of Swine Influenza Virus and Its Immunogenicity Analysis

作　　者：汪思钧[1] 廖明[1] 任涛[1] 张春红[2] 黄忠[2] 曹汉威[2] 

机构地区：[1]华南农业大学兽医学院,广州510642 [2]广东省农业科学院兽医研究所,广州510640

出　　处：《畜牧兽医学报》2007年第1期78-83,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技部农业成果转化资金项目(2005440060639);教育部留学回国人员科研启动基金(教外司留2006331);广东省科技计划项目(2005A20901003;2004B20201030;2005B20201010);广东省自然科学基金项目(5200638;05006241);广东省高教厅自然科学研究重点项目(粤财教[2005]126号)

摘　　要：通过细菌内同源重组的方法成功构建了表达H3N2亚型猪流感病毒血凝素的重组腺病毒。首先将血凝素基因亚克隆到穿梭载体质粒pAdenoVator-CMV5-IRES-GFP上,与腺病毒基因组质粒pAdenoVatorΔE1/E3共转化大肠杆菌BJ5183菌株,经细菌内同源重组产生重组腺病毒质粒pAd-HA-GFP。将该重组质粒转染293细胞以包装重组腺病毒。根据报告基因GFP的表达及Western-blot分析,证明获得了可正确表达猪流感病毒血凝素的重组腺病毒rAd-HA-GFP。重组腺病毒经一次性腹腔注射接种昆明鼠,2周后可检测到血凝抑制抗体,并且抗体至少可以持续12周,证实重组腺病毒介导表达的血凝素蛋白具有良好的免疫原性,可诱导小鼠产生高滴度的特异性抗体。A recombinant adenovirus expressing hemagglutinin （HA） gene of swine influenza virus （SIV） was constructed by homologous recombination technique. HA gene was sub-cloned into the vector pAdenoVator-CMV5-IRES-GFP. Then the shuttle plasmid was co-transformed into E. coli BJ5183 with adenovirus backbone plasmid pAdenoVatorAE1/E3 to generate a recombinant plasmid pAd-HA-GFP through homologous recombination. The plasmid was transferred into 293 cells for packing a recombinant adenovirus. The recombinant adenovirus expressing HA gene, named rAd-HA-GFP, was confirmed by the expression of green fluorescent protein （GFP） and western-blot analysis of HA protein. A single intraperitoneal injection （i. p. ） of rAd-HAGFP was conducted in Kunming mice, a high titer of HI antibody was detected at two weeks after the injection, and the antibody lasted at least for 12 weeks. The results showed that the HA protein expressed by rAd-HA-GFP possess a good immunogenicity.

关 键 词：猪流感病毒 血凝素基因 重组腺病毒 

分 类 号：S852.659.5[农业科学—基础兽医学]