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

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.