Gene cloning, sequencing, expression and biological activity of giant panda (Ailuropoda melanoleuca) interferon-alpha

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. In mammals, multiple subtypes of interferon-alpha (IFN-alpha) exist, most of which possess antiviral activity. Little is known about giant panda IFN-alpha genes and the role they may play in giant panda immunological responses to viruses. We have cloned genes encoding 12 giant panda IFN-alpha (AmIFN-alpha or AmIFNA) subtypes that share from 90 to 99% amino acid sequence identity. AmIFN-alpha12 has one additional amino acid at position 57, which is not present in other subtypes. Sequence identity of the AmIFN-alpha proteins encoded by the 12 genes compared to human IFN-alpha2 is approximately 58%. Unlike most of the human subtypes, each of the 12 giant panda IFN sequences has an N-glycosylation recognition site. Expression of all 12 AmIFN-alpha subtypes in 293 cells was confirmed by SDS-PAGE and Western blotting analysis. The antiviral activity and antiproliferative activity of each AmIFN-alpha subtype produced in transiently transfected 293 cell cultures were tested in vitro. All AmIFN-alpha subtypes were found to be stable at pH 2 or 65 degrees C and to exhibit antiviral activity. Some IFN subtypes (AmIFN-alpha8 and AmIFN-alpha4) showed higher biological activity levels than others, whereas AmIFN-alpha11 exhibited lower activity. AmIFN-alpha had various antiproliferative activities to different target cells. To B16 cells, AmIFN-alpha3, AmIFN-alpha4, AmIFN-alpha8 had the highest activities, while to K562 cells, AmIFN-alpha3, AmIFN-alpha7, AmIFN-alpha10 had the highest activities. The various IFN-alpha subtypes displayed a good correlation between their antiviral and antiproliferative potencies.