Molecular cloning and protein characterization of swine 4-1BB

Abstract

4-1BB is expressed on activated T cells and other immune and non-immune cells. It plays important roles in human and mouse T cell function. However, the swine 4-1BB sequence remains unknown and its role in swine T cell response has not been studied. In the present study, we for the first time described the cloning of the swine 4-1BB gene and the property of the protein. Two 4-1BB variants were detected in swine. The coding sequences of variant 1 and variant 2 were 768 and 726 nucleotides in length, respectively, and both variants were coded by 7 exons in the swine genome. Comparison of nucleotide and amino acid sequences showed that both swine 4-1BB variants were more closely related to bovine and human sequences than to either the mouse or rat sequence. Prediction analysis showed that swine 4-1BB belonged to the tumor necrosis factor receptor (TNFR) superfamily like human and mouse 4-1BB and the tertiary structures of the swine 4-1BB variants were much more similar to mouse 4-1BB than to human 4-1BB. The 1556bp 5′ regulatory sequence cloned by nested PCR efficiently induced green fluorescent protein expression in porcine peripheral blood mononuclear cells (PBMC) post nucleofection. Moreover, 4-1BB protein was widely expressed in pig tissues and both variants of swine 4-1BB protein were transmembrane proteins and expressed on the membrane of porcine PBMCs.