Cloning, Sequence Analysis, And Prokayotic Expression Of Galectin-3Cdna From Bufo Gargarizans And Bufo Japonicus Formosus

Galectin-3is a multifunctional β-galactoside binding protein, which has beenshown to play roles in diverse biological processes. With the antiapoptotic function intumor cells and the facilitating effect on the metastasis of tumors, galectin-3becomes anew target for cancer diagnosis, prognosis and treatment. Inhibitors of galectin-3havebeen considered to be a new type of anticancer drug candidates. As one of theseinhibitors, galectin-3C (N-terminal truncated galectin-3) is a secure and reliable one. Theobjective of the current study is to clone the cDNA of galectin-3from Bufo gargarizansand Bufo japonicus formosus, analyze the sequences by the aid of bioinformatics, andconstruct the recombinant plasmid for prokaryotic expression. The expression vector willbe a good foundation for further study on the biological activities of galectin-3C. Themain results are as follows:1. Five cDNA clones were obtained from the plasmid cDNA library of adult Bufojaponicus formosus skin (GenBank accession: JQ765589-JQ765593). To obtaingalectin-3cDNA from Chinese toad, B. gargarizans, a first-strand cDNA wassynthesized from total RNA of skin, which was used as the template for the subsequentPCR (polymerase chain reaction) amplification. The PCR products were successfullycloned into pGM-T vector and transformed into DH5α competent cells of E.coli. Aftersequencing, nine cDNA sequences of galectin-3were confirmed, one of which has beenregistered (X1-2GenBank accession: AEX58672.1). Three out of the fourteen cloneswere estimated to encode N-terminal truncated proteins (galectin-3C) naturally.2. The cDNA diversity and the deduced amino acid variation of B. gargarizansgalectin-3C were analyzed by the related softwares. High density of single nucleotidepolymorphism (SNP) was found,1SNP per20bp on average. Indentification of severalcDNA clones from one individual of two Bufo species might suggest the naturalexistance of the cDNA diversity of galectin-3, whose biological significance might bethe bio-adaptation to the variable habitats. Bioinformatical analysis showed that B.gargarizans galectin-3amino acid sequence contain two phosphorylation sites, whichplay important roles on its subcellular localization to the nuclei and cytoplasmicmembrane. galectin-3lacks amino-terminal signal peptide and transmembrane domain,which showed that the protein belonged to intracellular protein. Bufo galectin-3sharesphosphorylation sites, carbohydrate-binding sites and related motifs with mammaliangalectin-3including human, which suggests its carbohydrate-binding activity.3. Based on above analysis, specific primers were designed to construct recombinantpalsmids of either the full-length ORF of galectin-3or three different length ofgalectin-3C. The target cDNA segments were inserted into the prokaryotic expressionvector pET-28b(+), and their expression conditions were optimized.