The full-length of equine IFN-γ cDNA was cloned by RT-PCR and inserted into pCR2.1. The fragments were then cloned into the baculovirus transfer vector pAcYM1 (pAcEIFN-γ). The AcNPV or HyNPV DNA and pAcEIFN-γ were cotransfected to the SF21AE cells and the recombinant virus in transfected cell culture fluid was isolated. For production of the recombinant protein, TN5 cells and fifth-instar silkworm larvae were infected with the recombinant virus. When TN5 cells were infected with the virus, 14, 16, and 18 KDa proteins were detected by SDS-PAGE and Western blotting in the culture supernatants, and the supernatants contained high antiviral activity (1.3x106 U/ml). When infected cells were cultivated with tunicamycin, only 14 KDa proteins were detected and antiviral effects were decreased. So the reIFN-γ would be glycosylated form proteins and glycosylations were related to the biological activity. When silkworm larvae were inoculated with the virus, hemolymph collected from silkworm larvae was about 10 times higher (2.6x107 U/ml) than in both cell culture fluids. Several equine viruses were subjected for the antiviral tests. The reIFN-γ suppressed the replication of all equine viruses used in the present experiment in horse cell cultures. However, the antiviral effects were decreased in cells of heterologous species. High antiviral effect was observed especially against RNA viruses. Results from the present study show that reIFN-γ has a suppressing effect on equine viruses used in vitro. Thus, it is essential to find a practical use for reIFN in horses. (Viral Disease Section, Department of Infectious Diseases TEL +81-29-838-7841)
Reference:
Wu et al. (2002) Cytokine 20:63-69.
