Isolation, expression and bioactivity of feline granulocyte-macrophage colony-stimulating factor.
A cDNA encoding feline granulocyte-macrophage colony stimulating factor was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction (RT-PCR). The cDNA is 426 bp in length and encodes a predicted mature protein of 127 amino acids and the majority of the signal peptide. The recombinant protein (rfGM-CSF) was expressed in both Escherichia coli, as a calmodulin fusion protein, and mammalian cells. Biological activity of both recombinant proteins was demonstrated using the human erythroleukaemic cell line, TF-1. In a soft agar clonogenic assay, rfGM-CSF supported the development of granulocyte, macrophage and granulocyte-macrophage colonies. In combination with phytohaemagglutin (PHA) lymphocyte-conditioned medium, the number and size of such colonies were increased. Culture of feline bone marrow cells with rfGM-CSF was an efficient method for producing cells with morphology typical of dendritic cells (DC). The availability of the recombinant cytokine will permit further studies, in particular, the evaluation of the role of dendritic cells in feline immunopathology and its potential as a vaccine adjuvant.
Dunham
Stephen P
SP
Department of Veterinary Pathology, Retrovirus Research Laboratory, Institute of Comparative Medicine, University of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK. s.dunham@vet.gla.ac.uk
Bruce
Jennifer
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D016466:CHO Cells; D002415:Cats / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013936:Thymidine / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013936:Thymidine