Muneta Y, Shimoji Y, Inumaru S, Mori Y
National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
J Interferon Cytokine Res. 2001 May;21(5):305-12. doi: 10.1089/107999001300177493.
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.
我们分离并测序了包含猪Fas配体（FasL）编码序列的cDNA。基于人、小鼠和大鼠FasL中保守的5'和3'核苷酸序列，使用混合寡核苷酸引物，我们以用5μg/ml伴刀豆球蛋白A（ConA）刺激的猪胸腺细胞制备的总RNA进行逆转录聚合酶链反应（RT-PCR），以克隆猪FasL的cDNA。猪FasL cDNA的开放阅读框（ORF）长度为849个碱基对（bp），编码282个氨基酸。预测的氨基酸序列分别与人、小鼠和大鼠FasL的预测序列具有85.5%、76.6%和75.5%的同源性。由含有猪FasL完整编码序列的重组杆状病毒表达的重组猪FasL在经环己酰亚胺（CHX）致敏的猪肾小管细胞系PK-15细胞中显示出细胞毒性作用并诱导凋亡，分别通过MTT测定、DNA片段化测定和TUNEL染色得到证实。此外，猪白细胞介素-18（IL-18）可诱导猪外周血淋巴细胞（PBL）中猪FasL的mRNA表达。这些结果表明，本研究中鉴定的猪FasL具有生物学功能，并且具有与其他物种报道的一样诱导凋亡的能力。
