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Type: Journal article
Title: Cloning and expression of the ovine CD40 molecule and the inhibition of the mixed lymphocyte reaction by the ovine CD40e-EGFP fusion protein
Author: Zawitkowski, M.
Russ, G.
Krishnan, R.
Citation: Veterinary Immunology and Immunopathology, 2002; 89(1-2):37-45
Publisher: Elsevier Science BV
Issue Date: 2002
ISSN: 0165-2427
Abstract: The CD40 molecule is a member of the tumour necrosis factor receptor (TNFR)-like supergene family and plays a major role as a co-stimulatory molecule in the activation of T cells in response to antigens presented by dendritic cells. In this study, reverse transcription-PCR cloning was used to derive the sequence encoding ovine CD40. The ovine CD40 sequence demonstrated a similarity of 97, 76 and 64% with the bovine, human and murine sequences, respectively, at the nucleic acid level. The cysteine residues characteristic of the TNFR family and N-linked glycosylation sites are conserved. Furthermore, RNA analysis confirmed expression of CD40 mRNA in both ovine dendritic cells from lymphatic drainage and dermal fibroblasts in culture. In addition, cDNA encompassing the extracellular region of ovine CD40 (CD40(e)) was fused 'in-frame' with the enhanced green fluorescent protein (EGFP) to generate a fusion protein upon the transfection of Chinese hamster ovary (CHO) cells. Immunoprecipitation with an anti-GFP monoclonal antibody of a 78 kD a protein from conditioned medium of CHO transfectants confirmed that the CD40(e)-EGFP was secreted in the supernatant. All experiments were controlled with a pEGFP-N1 vector-blank construct. Moreover, the biological activity of ovine CD40(e)-EGFP was demonstrated by its ability to inhibit a two-way mixed lymphocyte reaction. Thus these observations confirm that ovine CD40 blockade inhibits co-stimulation mediated by CD40-CD40L (CD154) interactions as has been reported in murine and human studies.
Keywords: Jurkat Cells; CHO Cells; Animals; Sheep; Humans; Luminescent Proteins; Green Fluorescent Proteins; Recombinant Fusion Proteins; RNA; Lymphocyte Culture Test, Mixed; Precipitin Tests; Cloning, Molecular; Transfection; Reverse Transcriptase Polymerase Chain Reaction; Amino Acid Sequence; Base Sequence; Molecular Sequence Data; Cricetinae; CD40 Antigens
RMID: 0020020191
DOI: 10.1016/S0165-2427(02)00182-4
Appears in Collections:Medicine publications

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