McGlothlin James R, Gao Li, Lavoie Tera, Simon Brett A, Easley R Blaine, Ma Shwu-Fan, Rumala Bernice B, Garcia Joe G N, Ye Shui Qing
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Mason F. Lord Memorial Building, Center Tower, Rm. 665, 5200 Eastern Avenue, Baltimore, Maryland 21224, USA.
Biochem Genet. 2005 Apr;43(3-4):127-41. doi: 10.1007/s10528-005-1505-2.
During our previous attempt to search for the candidate genes to acute lung injury (ALI), we unexpectedly identified PBEF as the most highly upregulated gene in a canine model of ALI by crosshybridizing canine lung cRNA to the Affymetrix human gene chip HG-U133A. The result suggested that PBEF may be a potential biomarker in ALI. To extend and translate that finding, we have performed the molecular cloning and characterization of canine PBEF cDNA in this study. Deduced amino acid sequence alignment revealed that the PBEF gene is evolutionarily highly conserved, with the canine PBEF protein sequence 96% identical to human PBEF and 94% identical to both murine and rat PBEF counterparts. Canine PBEF protein was successfully expressed both by in vitro transcription coupled with translation in a cell-free system and by transfection of canine PBEF cDNA into the human lung type II alveolar adenocarcinoma cell line A549. The expressed canine PBEF protein was visualized by either an anti-V5 tag peptide polyclonal antibody or an anti-canine PBEF peptide polyclonal antibody. RT-PCR assay indicates that canine PBEF is expressed in canine lung, brain, heart, liver, spleen, kidney, pancreas, and muscle, with liver showing the highest expression,followed by muscle. Isolation of the canine PBEF cDNA and expression of its recombinant protein may provide molecular tools to study the molecular mechanism of ALI in the canine model and to elucidate the potential role of PBEF as an ALI biomarker.
在我们之前寻找急性肺损伤（ALI）候选基因的尝试中，通过将犬肺cRNA与Affymetrix人类基因芯片HG-U133A进行交叉杂交，我们意外地发现PBEF是ALI犬模型中上调程度最高的基因。这一结果表明，PBEF可能是ALI的一个潜在生物标志物。为了扩展并转化这一发现，我们在本研究中进行了犬PBEF cDNA的分子克隆和特性分析。推导的氨基酸序列比对显示，PBEF基因在进化上高度保守，犬PBEF蛋白序列与人类PBEF的一致性为96%，与小鼠和大鼠的PBEF对应序列的一致性均为94%。犬PBEF蛋白通过体外转录结合无细胞系统中的翻译以及将犬PBEF cDNA转染到人肺II型肺泡腺癌细胞系A549中均成功表达。通过抗V5标签肽多克隆抗体或抗犬PBEF肽多克隆抗体均可观察到表达的犬PBEF蛋白。RT-PCR分析表明，犬PBEF在犬的肺、脑、心脏、肝脏、脾脏、肾脏、胰腺和肌肉中均有表达，其中肝脏表达最高，其次是肌肉。犬PBEF cDNA的分离及其重组蛋白的表达可能为研究犬模型中ALI的分子机制以及阐明PBEF作为ALI生物标志物的潜在作用提供分子工具。
