Fusion Genes Ncmag1of Bovine Neospora Caninum And IFN-гGENE Was Expressed And Was Immunological Evaluated

Neosporiasis is a kind of protozoosis which parasitic on a variety of animal cells by Neospora caninum. N. caninum is a top composite class parasitic protozoa. It mainly causes the abortion of pregnant cows, stillbirth and nervous system disorders. Currently no effective drugs and vaccines can be used to prevent and control the prevalence of the disease.Based on the published N. caninum NcMAG1gene sequence (GenBank accession number:EF580924.1) and IFN-γ gene sequence (GenBank accession number:M29867.1), designed and synthesized two pairs containing specific primers,which were used for prokaryotic and eukaryotic expression. Bovine N. caninum NcMAG1, bovine IFN-γ gene fragment were amplified by PCR. The fragment was cloned into pMD18-T simple vector and then cloned into pGEX-4T-1prokaryotic expression vector and pVAXI eukaryotic expression vector, respectively. The correct fusion expression plasmids were used for prokaryotic and eukaryotic expression. Finally, the purified NcMAG1-IFN-gamma fusion protein subunit vaccines and NcMAG1-IFN-gamma fusion gene DNA vaccine immunized to the BALB/c mice and detected the level of humoral immunity and cellular immunity levels by the application of indirect ELISA and flow cytometry.The results showed that constructed the prokaryotic expression plasmid pGEX-NcMAG1-IFN-γ containing the NcMAG1-IFN-γ and eukaryotic expression plasmid pVAX-NcMAGl-IFN-γ-containing fusion gene NcMAG1-IFN-γ successfully. Prokaryotic expression products were identified by SDS-PAGE and western-blotting, proved that the fusion protein was82ku in size; The integration of recombinant plasmid was successfully transfected into Vero cells by IFA and Western-blotting, and the protein expressed57ku in size and have good reactogenicity. NcMAGl-IFN-γ fusion protein subunit vaccine and pVAX-NcMAG1-IFN-γ DNA vaccine could induce BALB/c mice to produce a high level of humoral immunity and cellular Immunity. This test opens up a new way for N. caninum DNA vaccine development.