Bufo Transgelin-2cDNA Cloning And Bioinformatic Analysis As Well As Its Antiserum Preparation

Bufo-origin materials have long been used in traditional Chinese medicines and many studieshave indicated abundant bioactive polypeptides included in Bufo skin and their anti-tumor curativeeffects. The objective of the current study is to clarify the bioactive polypetides included inBufo-origin materials by cloning cDNA sequences from Chinese toad B. gargarizans and Japanesetoad B. japonicus formosus. Then the the correponding recombinant plasmids for prokaryoticexpression were constructed, whose recombinant protein were used as antigen to make anti-mouseantiserum of transgelin-2(TAGLN2). The main results are as follows:1. By colony PCR (polymerase chain reaction), a cDNA sequenceof TAGLN2(GenBankaccession number: JX197456) were obtained from adult Japanese toad skin plasmid cDNA librarywith a deletion including the whole5′-untranslated region (UTR) and partial open reading frame(ORF). The transcript is997bp consisting of31bp5′-UTR,618bp3′-UTR and an ORF of348bpencoding a polypeptide of115amino acids. Homologous analysis indicated the similarity ofJapanese toad TAGLN2was71%-85%with those of other species.2. To confirm the expression of the full length TAGLN2in Japanese toad, two primers (P1and P2) were designed based on the partial TAGLN2sequence, and two PCR (polymerase chainreaction) reactions (P1and XhoTT, and P2and SP6as a pair of primers, respectively) wereperformed to clone the5′-UTR and3′-UTR using the same skin cDNA library as the template, andthe PCR products were cloned. Based on the newly cloned TAGLN25′-UTR and3′-UTRsequences, two new primers (P3and P4) were designed and the second round PCR was performedby pairing P3with XhoTT and P4with SP6using Japanese toad skin cDNA library as thetemplate. As the result,28clones of TAGLN2cDNAs were obtaied, and the longest transcript(GenBank accession number: KC820703) is1267bp consisting of86bp5′-UTR and587bp3′-UTR and an ORF of594bp encoding a polypeptide of197amino acids. Other clones showeddifferent length of5′-UTR, some of which even lost their partial ORF sequence. Meanwhileseveral single nucleotide polymorphism (SNP) sites and some deletions and insertions wereindicated. All these results indicated TAGLN2gene diversity. However, their encoding proteins allhave the same amino acid sequences. Homologous analysis of Japanese toad full length TAGLN2 showed the similarity of71%-78%with those of other species.3. For the further exploration of Bufo, authors cloned TAGLN2cDNA from Chinese toadskin first strand cDNAs (GenBank accession number: KF432856). The transcript is1207bpconsisting of16bp5′-UTR,594bp3′-UTR and an ORF of597bp encoding a polypeptide of197amino acid residues. Only one amino acid residue is different between Japanese toad TAGLN2and Chinese one. RT-PCR experiments showed that TAGLN2expressed in all Chinese toad organstested so far, while the expression in lung, liver and kidney was more obvious than in otherorgans.4. The recombinant plasmid of Japanese toad TAGLN2was made, whose recombinantprotein was induced, purified and used as the antigen to immune Balb/c mouse to prepareanti-mouse TAGLN2antiserum. Indirect enzyme linked immunosorbent assay (ELISA) indicateda titer of1:60000of the antiserum, which showed excellent specificity tested by western blot.Anti-TAGLN2antiserum will provide a reference for the further studies on its biological functionsand on the expression of TAGLN2in toad organisms.