表达H5N1亚型猪流感病毒HA基因重组腺病毒的构建及其免疫原性  

Construction and Immunogenicity of a Recombinant Adenovirus Expressing HA Gene of H5N1 Subtype Swine Influenza Virus

作　　者：吴运谱[1,2] 乔传玲[1] 杨焕良[1] 陈艳[1] 展小过[1] 辛晓光[1] 陈化兰[1] 

机构地区：[1]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农业部动物流感重点开放实验室,哈尔滨150001 [2]辽宁省动物疫病预防控制中心,沈阳110164

出　　处：《畜牧兽医学报》2009年第9期1363-1369,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技攻关计划项目(2004BA519A55);国家重点实验室科研业务费项目(NKLVBP200818)

摘　　要：RT-PCR扩增猪流感病毒A/Swine/Fujian/1/2001(H5N1)株的HA基因,构建重组腺病毒穿梭质粒pDC315-H5 HA-EGFP。采用Ad-Max同源重组系统和共转染技术,构建了表达H5N1亚型猪流感病毒HA基因的复制缺陷型重组腺病毒(rAd-H5 HA-EGFP)。经目的基因PCR检测及序列测定,结果表明:HA基因已经正确地插入到腺病毒的基因组中;通过RT-PCR检测与Western blot分析,结果表明:HA基因能够进行正确转录,并且所表达的蛋白具有相应的生物学活性。子代重组腺病毒rAd-H5 HA-EGFP经增殖、纯化后感染性滴度可达2.26×1010TCID50mL-1。rAd-H5 HA-EGFP免疫BALB/c小鼠能够诱导特异性的HI抗体产生,有效阻止病毒在体内的复制。To construct recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,HA gene of A/Swine/Fujian/1/2001（H5N1） was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing HA gene（rAd-H5HA-EGFP） was generated by co-transfecting HEK293 cells with the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cell with natural biological activities.The TCID50 of the rAd-H5HA-EGFP was evaluated as 2.26×10^10 mL^-1 after propagation and purification.And the results of immunization in BALB/c mice indicated rAd-H5HA-EGFP could induce HI antibodies and inhibit virus replication in mice lungs.

关 键 词：猪流感病毒 H5N1亚型 HA基因 重组腺病毒 

分 类 号：S852.659.5[农业科学—基础兽医学]