| Based on the status quo of China’s aquatic animal diseases, the study ofinfectious hematopoietic necrosis virus (BJLL strain) isolated from diseased fishfarms within Beijing was carried out in our laboratory. Cell culture, identificationof this virus and its biological characteristics were performed. On this basis,mouse anti-IHNV polyclonal antibody was prepared, IHNV N and G gene wascloned and expressed in baculovirus expression system, and the two proteins weretested by Western-blot and IFA. Using purified N protein as immunogen, mouseantiserum was prepared, and then the immunological activities of the N proteinwas tested. The results are as follows:1Proliferation and purification of IHNV and anti-virus serum preparationIHNV was proliferated in Chinook salmon embryo cell line (CHSE). The titerof the IHNV and the growth curve were determined. The IHNV in cell culture washarvested and purified using ultracentrifugation. Balb/c mice were immunizedwith purified virus to prepare polyclonal antibody, and mouse anti-IHNV serumwas titrated as1:8000using indirect ELISA assay.2Expression, purification and antigenic analysis of IHNV N proteinAccording to the published sequence of IHNV N gene, a pair of oligonucleotideprimers was designed, the N gene was obtained from the genomic cDNA of IHNVby PCR. This Gene was cloned into pGM-T vector, and the recombinant plasmidwas identified by PCR and sequencing. The results showed that the nucleotidesequence of amplified N gene has99％homology with the reported N genesequences. In this study nucleoprotein (N) gene(1176bp) was amplified byRT-PCR from IHNV and inserted into the baculovirus vector pFB-LIC-Bse toconstruct a recombinant plasmid pFB-LIC-Bse-N. The constructed recombinanttransposition plasmid pFB-LIC-Bse-N was transformed to competent E. coliDH10Bac to get recombinant bacmids reBacmid-N. The obtained recombinantbacmid reBacmid-N was transfected to insect Sf9cells cultured at27℃,then cellprecipitation was collected after72h～96h. The expressed protein was analyzedby SDS-PAGE electrophoresis, the size was consistent with the expectancy.Western-blot and IFA analysis showed that the expressed protein had a good immunogenicity. Balb/c mice were immunized with purified N protein to preparehyperimmune serum. N protein serum titer was measured using purified viruscoated ELISA plates and mouse anti-N protein serum titer was1:8000.3Expression and identification of the G gene of IHNVAccording to the published sequence of IHNV G gene, a pair of oligonucleotideprimers was designed, the G gene was obtained from the genomic cDNA of IHNVby PCR．This Gene was cloned into pGM-T vector, and the recombinant plasmidwas identified by PCR and sequencing. In this study Glycoprotein (G) gene(1380bp) was amplified by RT-PCR from IHNV and inserted into the baculovirus vectorpFB-LIC-Bse to construct a recombinant plasmid pFB-LIC-Bse-G. Theconstructed recombinant transposition plasmid pFB-LIC-Bse-G was transformedinto competent E. coli DH10Bac to get recombinant bacmids reBacmid-G. Theobtained recombinant bacmid reBacmid-G was transfected to insect Sf9cellscultured at27℃, cell precipitation was collected after72h～96h. The expressedprotein was analyzed by SDS-PAGE electrophoresis, the size was consistent withthe expectancy. Western-blot and IFA analysis showed that the expressed proteinhad a good immunogenicity．... |
