Submitted to: Nucleic Acids Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 25, 1994
Publication Date: N/A
Interpretive Summary: Interleukin 12 (IL12) is 70 kDa heterodimeric cytokine important in the growth and differentiation of T lymphocytes. As such, it may play a major role in cell mediated immunity directed against infectious agents and tumors. Because of the likelihood of species specificity among cytokines, it is important to study the homologous subunits of IL12 to assess their function(s) in cattle responding to infection. We describe here, the molecular cloning of the 40 kDa subunit of bovine IL12.
Total RNA was isolated from cultured bovine peripheral blood mononuclear cells following a 16 h stimulation with calcium ionophore A23187 (1 ug/ml) and phorbol 12-myristate 13-acetate (5 ng/ml). cDNA synthesis was performed using oligo dT as primer. Initial polymerase chain reaction primers were generated from partially conserved regions upstream from the translation start site and downstream from the translation stop sight of human (1) and mouse (2) IL12 40 kDa subunit sequences. cDNA amplification was initially performed at low primer annealing temperatures (37 C; 60 sec) followed by agarose gel size fractionation to generate sufficient material for preliminary sequence data. Specific primers at the 5' and 3' ends of the open reading frame were then synthesized with Bam HI and Hind III restriction sites, respectively, to allow reamplification of the entire open reading frame directly from cDNA and directional cloning into pUC 18 plasmid DNA. Several clones were sequenced for verification. The 984 base pair open reading frame of BIL1240 possesses a single Eco RI site at base 744 and shows 87.8% and 75.6% sequence homology with human (1) and mouse (2) sequences while exhibiting 84.4% and 67.6% similarity at the predicted amino acid level, respectively.