Cloning, expression, purification, and biological activity of five feline type I interferons.
Type I interferons (IFN) are important mediators of the host defense against viral infections in mammals. In humans multiple subtypes of IFN-alpha exist, most of which possess antiviral activity. Little is known about the type I IFN genes in cats and the role they may play in feline immunological responses to viruses. We have isolated cDNAs encoding five feline IFN-alpha (feIFN) subtypes that share from 95 to 99% amino acid sequence identity. FeIFN-alpha5 has five additional amino acids inserted at position 139, which are not present in the other four subtypes. Sequence identity of the feIFN proteins encoded by the five clones compared to human IFN-alpha2 is approximately 60%. Unlike most of the human subtypes, each of the five feline IFN sequences has an N-glycosylation recognition site. Expression of all five feIFN-alpha subtypes in Chinese hamster ovary (CHO) cells was confirmed by Western blot analysis, and all resulting proteins were glycosylated. The antiviral activity of each feIFN-alpha subtype produced in transiently transfected CHO cell cultures was tested in vitro. In addition, subtype feIFN-alpha6 was expressed in the yeast, Pichia pastoris. The resulting secreted mature recombinant protein was purified and demonstrated significant antiviral activity and induction of 2',5'-oligoadenylate synthetase activity in vitro.
Wonderling
Ramani
R
Heska Corporation, 1613 Prospect Parkway, Fort Collins, CO 80525, USA.
Powell
Tim
T
Baldwin
Susan
S
Morales
Tony
T
Snyder
Scott
S
Keiser
Kathy
K
Hunter
Shirley
S
Best
Elaine
E
McDermott
Martin J
MJ
Milhausen
Michael
M
D015088:2',5'-Oligoadenylate Synthetase / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D016466:CHO Cells; D002415:Cats / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007370:Interferon Type I / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014721:Vesicular stomatitis Indiana virus / Q000276:immunology
D007370:Interferon Type I; D011994:Recombinant Proteins; D012313:RNA; D015088:2',5'-Oligoadenylate Synthetase