H1亚型猪流感病毒血凝素基因在昆虫细胞中的表达及其间接ELISA方法的初步建立  

Baculovirus expression and establishment of the indirect ELISA for the HA gene of swine influenza virus H1 subtype

作　　者：万春和[1] 刘明[1] 刘春国[1] 张晓霁[1] 杨涛[1] 刘大飞[1] 陈浩[1] 齐金龙[1] 乔传玲[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,哈尔滨150001

出　　处：《微生物学报》2008年第2期220-225,共6页Acta Microbiologica Sinica

基　　金：国家“973项目”(2005CB523202);国家“十一五”科技支撑计划项目(2006BAD06A05)~~

摘　　要：根据GenBank发表的H1亚型猪流感HA基因序列设计引物,扩增出HA基因片段,将其克隆到pFastBacGP67B杆状病毒载体上,筛选阳性重组转座载体pFastBacGP67B-H1,转化含有杆状病毒穿梭载体(bacmid)的DH10Bac感受态细胞,构建杆状病毒表达载体获得重组转座子(rBacmid-H1),在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒(rBV-H1),再感染细胞,收获目的蛋白。通过血凝试验、免疫印迹法、免疫组化分析表明该蛋白得到表达,且具有良好的生物学活性。利用表达的蛋白作为猪流感间接ELISA的抗原,初步建立H1亚型猪流感的间接ELISA检测方法,并对内蒙古、辽宁和黑龙江等地送检的93份猪血清进行了检测,阳性率为31.18%,为研制开发快速、准确、简便的H1亚型猪流感鉴别诊断试剂盒奠定基础。The hemagglutinin （HA） gene fragment of swine influenza virus A/Swine/Guangdong/LM/05（HIN1） was amplified with HA gene specific primers and cloned into baculovirus transfer plasmid pFASTBacGP67B. The recombinant plasmid pFastBacGP67B-H1 was identified by restriction enzyme digestion and gene sequencing. Following the transformation of DHIOBac Escherichia coli component cells by pFastBacGP67B-H1, recombinant bacmids rBacmid-H1 were identified by blue/white selection and PCR analysis. Then recombinant baculovirus rBV-H1 was rescued by lipofectant reagent Cellfectin induced rBacmid-H1 DNA transfection of long-phage sf9 insect cells. The recombinant HA protein was characterized by hemagglutination test, western-blot and immunohistochemistry. An indirect enzyme-linked immunosorbent assay （ELISA） was assessed to detect in pigs IgG against H1 subtype SIV present in Inner Mongolia, Liaoning and Heilongjiang provinces. Positive was found in 31.15% （29 of 93） serum samples tested from swine reared in commercial herds. However, all irrelevant control sera tested were negative. We conclude, therefore, that ELISA performed with recombinant HA as coating antigen was a better tool for swine influenza surveillance in China.

关 键 词：H1亚型猪流感病毒 血凝素基因 昆虫细胞杆状病毒表达 间接ELISA 

分 类 号：S854.43[农业科学—临床兽医学]