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

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.

Key words:H1 subtype SIV; HA gene; baculovirus/insect cell system; indirect ELISA