| Bovine Interferon-gamma (BoIFN-γ) has the characteristics of virustatic, antitumor and immunoloregulation function. This study succeeded in cloning, expression and purification of the recombinant Bovine IFN-γfusion protein, and preparation of rabbit polyclonal antibody against BoIFN-γ. This provides a solid foundation for further research on BoIFN-γbiological properties and its applications.Bovine spleen lymphocytes were stimulated with ConA, then total RNA was extracted and used as template to amplify the IFN-γgene with the method of RT-PCR. The amplified fragment was subsequently ligated into the pMD18-T vector for sequencing. The results revealed that the IFN-γgene is 498bp longth, which can encode 166 amino acid. Comparison of the sequenced IFN-γwith the reported BoIFN-γsequence in GenBank revealed 100% identity at the nucleotide level. And shared 81.3%, 74.9%, 73.6%, 53.7%, 50.7% identities with goat, pig, human, mice, chicken IFN-γgene in nucleotide, respectively.The BoIFN-γgene was subcloned into an expression vector pET-30a (+) and constructed the recombinant expression plasmid of pET-BoIFN-γ. Then it was transformed into E.coli BL21 cells, and induced by 0.5mmol/L IPTG at 37℃for 6 hours. The recombinant BoIFN-γwas expressed efficiently in the form of fusion protein with the yield accounting for 33% of total bacterial proteins. SDS-PAGE and Western blot analysis showed that the recombinant fusion protein had a molecular weight of approximate 25ku. After purification, the target protein was harvested with a concentration of 1.6mg/ml. And the polyclonal antibody against BoIFN-γwere generated by immunizing rabbits with the purified protein. The AGP and western blot experiment showed that the antibody could react with purified recombinant BoIFN-γprotein.
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