两种蟾蜍EDF-1 cDNA的克隆及其生物信息学分析

Cloning and its bioinformatic analysis of EDF-1 cDNA from two Bufo species

蟾蜍皮肤基源的中药材（如蟾皮和蟾酥）,已长期应用于临床,主要治疗炎症、疼痛以及多种肿瘤。为分析这些蟾蜍中药材中含有的多肽类有效成分,通过菌落DNA聚合酶链式反应（colony polymerase chain reaction）对日本蟾蜍（Bufo japonicus formosus）皮肤cDNA质粒文库进行筛选,克隆到958 bp的转录子（GenBank accession number：KF769458）,包括5＇端13 bp、3＇端498 bp的非翻译区和447 bp的开放阅读框（open reading frame,ORF）,编码由148个氨基酸残基组成的蛋白质,与人及其他动物的内皮分化相关因子-1（endothelial differentiation-related factor-1,EDF-1）显示高度同源性。为分析中华大蟾蜍（B.gargarizans）皮肤中是否存在EDF-1的表达,在日本蟾蜍cDNA序列的基础上设计引物,并以中华大蟾蜍皮肤第一链cDNA为模板,通过PCR克隆中华大蟾蜍EDF-1 ORF。测序分析表明其ORF为447 bp（GenBank accession number：KF769459）,与日本蟾蜍的ORF长度相同,两者编码的蛋白之间存在3个氨基酸的差异,但与人源EDF-1的相似性均为95%,与其他动物的相似性介于93%-97%,表明其在进化上的高度保守性。磷酸化位点预测显示9个潜在的磷酸化位点,与已报道的该蛋白的生物学活性受上游因子的调控的研究结果相吻合。由于哺乳动物的EDF-1参与抑制内皮细胞的分化,因此它很可能是蟾蜍皮肤基源的中药材中含有的抗肿瘤的有效成分之一。

In traditional Chinese medicine, toad skin-origin materials including toad skin and its secretions have long been used to treat inflammation, sores and even many kinds of cancers. To clarify the polypeptides included in these toad-origin materials, screening of cDNAs encoding polypeptides was carried out from the plasmid skin cDNA library of Japanese toad （ Bufo japonicus formosus） by colony polymerase chain reaction （ PCR）. As the result, a transcript of 958 bp （GenBank accession number： KF769458） was obtained consisting of 13 bp 5＇ untranslated region （UTR）, 498 bp 3＇ UTR and an open reading frame （ORF） of 447 bp encoding a polypeptide of 148 amino acid residues with high homology with endothelial differentiation-related factor-1 （EDF-1） of other animals. Based on the EDF-1 cDNA sequence of Japanese toad, a pair of primers were designed for cloning Chinese toad （ B. gargarizans） EDF-1 and a DNA segment was obtained with an ORF of 447 bp （ GenBank accession number： KF769459） encoding a polypeptide of 148 amino acids, among which three amino acid residues were different from that of Japanese toad. For two Bufo species, the similarity is 95% with human EDF-1 and 93% -97% with other animals indicating that EDF-1 is evolutionarily highly conserved. Besides, phosphorylation site prediction indicated 9 potential sites suggesting its activity being upstream regulated which is consistent with previous reports. Due to the involement in the process of endothelial cell differentiation, EDF-1 expressed in toad skin might be one of the important ingredients with antitumor effects.