Wan Chunhe, Liu Ming, Liu Chunguo, Zhang Xiaoji, Yang Tao, Liu Dafei, Chen Hao, Qi Jinlong, Qiao Chuanling
Animal Influenza Laboratory of the Ministry of Agriculture, Harbin Veterinary Research Institute of CAAS, Harbin 150001, China.
Wei Sheng Wu Xue Bao. 2008 Feb;48(2):220-5.
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 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.
用甲型流感病毒A/猪/广东/LM/05(H1N1)的血凝素(HA)基因特异性引物扩增HA基因片段，并克隆到杆状病毒转移质粒pFASTBacGP67B中。通过限制性内切酶消化和基因测序鉴定重组质粒pFastBacGP67B-H1。用pFastBacGP67B-H1转化DH10Bac大肠杆菌感受态细胞后，通过蓝白筛选和PCR分析鉴定重组杆粒rBacmid-H1。然后用脂质体转染试剂Cellfectin诱导rBacmid-H1 DNA转染长噬菌体sf9昆虫细胞，拯救重组杆状病毒rBV-H1。通过血凝试验、western-blot和免疫组织化学对重组HA蛋白进行鉴定。评估了一种间接酶联免疫吸附试验(ELISA)，以检测内蒙古、辽宁和黑龙江三省猪血清中针对H1亚型猪流感病毒的IgG。在商品猪场饲养的猪的93份检测血清样本中，有31.15%(29份)呈阳性。然而，所有检测的无关对照血清均为阴性。因此，我们得出结论，以重组HA作为包被抗原的ELISA是中国猪流感监测的更好工具。
