机构地区: 华南农业大学兽医学院农业部动物疫病防控重点开放实验室
出 处: 《中国预防兽医学报》 2009年第5期333-336,355,共5页
摘 要: 本研究对猪流感病毒A/Swine/Guangdong/3/2003(H1N1)株(SIV-GD03株)的HA基因序列进行了遗传演化分析,结果表明SIV-GD03株与GenBank中登录的全部192株H1亚型SIV中的151株核苷酸序列相似性达到85%以上,表明该毒株具有作为疫苗候选株的分子特征。将SIV-GD03的HA基因克隆到腺病毒穿梭载体pAd-CMV5-IRES-GFP中,将得到的重组穿梭质粒pAd-H1-IRES-GFP电转化至BJ5183感受态细胞中,与人5型腺病毒骨架质粒同源重组获得重组腺病毒质粒prAd-H1-IRES-GFP,该质粒线性化后转染AD-293细胞。包装出的重组腺病毒rAd-H1HA-GFP通过荧光显微镜观察、PCR和western blot鉴定,证明HA基因和GFP基因在重组腺病毒中得到高效表达,该毒株在AD-293细胞上连续传12代后表现出较好的整合稳定性及传代稳定性。 The hemagglutin (HA) gene of H1 subtype swine influenza virus (SIV-GD03) was cloned into the adenovirus vector and transformed into E.coli BJ5183 to generate a recombinant plasmid. The recombinant plasmid was transfected into AD-293 cells for packing a recombinant adenovirus of rAd-H1HA-GFP which was confirmed by PCR and western blot analysis. rAd-H1HA-GFP was passaged serially for 12 passages in AD-293 cells. The results indicated that rAd-H1HA-GFP was stable through PCR identification and TCID50 determination.