Cloning, expression of human IL 18 cDNA and the effect of chemical stimuli on IL 18 mRNA expression
G.W. Wong1, A.M. Ali2 and H.F. Sew1*
1Immunology Unit, Department of Clinical Laboratory Science, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
2Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia
3400 Serdang, Selangor, Malaysia
(Received 23 March 1999 /Accepted 15 August 1999)
Abstract.
Human IL-18 cDNA which was amplified via the reverse transcription polymerase chain reaction of total RNA isolated from KU812F, a chronic myelogenous leukemic cell line, was successfully cloned into pTrcHis-2TOPO vector and expressed in E. coli. The authenticity of the human IL-18 cDNA sequence was confirmed by DNA sequencing. It exhibited 100% identity to the human IL-18 cDNA sequence as published in the database Genbank (accession no. D49950). SDS PAGE analysis of the crude lysates from pTrcHis TOPO.1L-18 cultures induced with I mM IPTG yielded a prominent protein band of the expected molecular mass of approximately 23kDa. The expressed recombinant protein was purified from the soluble fraction using the Talon metal affinity resin. A human T cell line, CEM-SS and PBMC were shown to exhibit constitutive basal levels of IL-18 mRNA expression. Con A and PMA upregulated IL-18 mRNA expression in CEM SS. Dibutyryl cyclic AMP and dexamethasone were found to downregulate IL-18 mRNA synthesis in PBMC.
Keywords: 11,18, cloning, expression, pTrcHis2 TOPO
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