禽流感病毒H5亚型HA基因在杆状病毒系统中的表达及生物学活性鉴定  

Expression of HA gene of avian influenza virus in baculovirus expression system and its bio-activity identification

作　　者：赵双成[1] 常晓飞[1] 田石[1] 柳金雄[1] 陈普成[1] 田国彬[1] 邓国华[1] 姜永萍[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农业部动物流感重点开放实验室/兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2012年第7期523-526,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：哈尔滨市科技攻关计划项目(2011AA6BN019)

摘　　要：为研究禽流感病毒(AIV)HA蛋白抗原活性,本研究将A/Goose/Guangdong/1/96(H5N1)的HA基因片段克隆于pFastBacHTA中,构建重组质粒pFastBacHTA-HA,转化至DH10Bac感受态细胞,构建重组杆粒,在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒。采用间接免疫荧光、western blot和血凝试验对所表达的蛋白进行抗原性和生物学活性分析。结果表明,表达重组蛋白分子量约为64 ku;IFA和western blot试验证明重组HA蛋白能够与H5亚型禽流感阳性血清特异结合,具有良好的抗原性;血凝试验和淋巴细胞增殖试验显示重组蛋白具有良好生物学活性。重组蛋白的正确表达为进一步研究HA基因功能活性和制备禽流感HA基因亚单位疫苗奠定了基础。For eukaryotic expression of avian influenza virus （AIV） hemagglutinin （HA）, the HA gene of A/Goose/ Guangdong/1/96（H5N1） was cloned into the baculovirus transfer vector pFastBacHTa and the resultant recombinant plasmid was transformed into DH10Bac competent cells to generate recombinant bacmid （rBacmid-HA）, which was identified by PCR and sequencing. The recombinant baculovirus stock was prepared by transfecting rBacmid-HA into the Sf9 insect cells. The expression of the recombinant HA in St9 cells was identified by indirect immunofluorescence assay and westem blot, which shown that the recombinant HA were about 64 ku. Further identifications demonstrated that the HA was able to induce high titer of HI antibody and effectively stimulate proliferation of lymph cells in peripheral blood of SPF chicken detected by MTT assay. The preparation of the recombinant protein in S19 cells was facilitated the further study of HA fimctions and preparation of the subunit vaccine against AIV infection.

关 键 词：禽流感 HA基因 杆状病毒 

分 类 号：S852.65[农业科学—基础兽医学]