【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。更多还原

【Abstract】 The hemagglutinin(HA)gene fragment of swine influenza virus A/Swine/Guangdong/LM/05(H1N1)was amplified with HA gene specific primers and cloned into baculovirus transfer plasmid pFASTBacGP67B.The recom- binant plasmid pFastBacGP67B-H1 was identified by restriction enzyme digestion and gene sequencing.Following the transformation of DH10Bac Escherichia coli component cells by pFastBacGP67B-H1,recombinant bacmids rBac- mid-H1 were identified by blue/white selection and PCR analysis.Then recombinant baculovirus rBV-H1 was rescued by lipofectant reagent Cellfectin induced rBacmid-H 1 DNA transfection of long-phage sf9 insect cells.The recombinant HA protein was characterized by hemagglutination test,western-blot and immunohistochemistry.An indirect en- zyme-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.更多还原