Molecular cloning, characterization, and expression of porcine Fas ligand (CD95 ligand).
We isolated and sequenced cDNA that contained the coding sequence of porcine Fas ligand (FasL). Using mixed oligonucleotide primers based on the 5' and 3' nucleotide sequences conserved among human, murine, and rat FasL, we performed the reverse transcription polymerase chain reaction (RT-PCR) with total RNA prepared from porcine thymocytes stimulated with 5 microg/ml concanavalin A (ConA) to clone the cDNA of porcine FasL. The open reading frame (ORF) of porcine FasL cDNA was 849 base pairs (bp) in length and encoded 282 amino acids. The predicted amino acid sequence was 85.5%, 76.6%, and 75.5% homologous to the predicted human, murine, and rat FasL, respectively. The recombinant porcine FasL expressed by recombinant baculovirus containing the whole coding sequences of porcine FasL showed cytotoxic effect and induced apoptosis in porcine renal tubular cell line PK-15 cells sensitized by cycloheximide (CHX), which was confirmed by MTT assay, DNA fragmentation assay, and TUNEL staining, respectively. Furthermore, the mRNA expression of porcine FasL in porcine peripheral blood lymphocytes (PBL) was induced by porcine interleukin-18 (IL-18). These results indicate that porcine FasL identified in this study is biologically functional and has the ability to induce apoptosis as reported in other species.
Muneta
Y
Y
National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. ymuneta@niah.affrc.go.jp
Shimoji
Y
Y
Inumaru
S
S
Mori
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D053938:DNA Fragmentation / Q000235:genetics / Q000276:immunology; D053222:Fas Ligand Protein; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020287:In Situ Nick-End Labeling; D020382:Interleukin-18 / Q000502:physiology; D008024:Ligands; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D013552:Swine; D015854:Up-Regulation / Q000276:immunology; D019014:fas Receptor / Q000378:metabolism*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; C505662:Faslg protein, rat; D020382:Interleukin-18; D008024:Ligands; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019014:fas Receptor