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

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

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。

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 recombinant 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-H1 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.