Cloning Of MCL-1cDNA From Two Bufo Species And Their Recombinant Protein Expression As Well As Antiserum Preparation

Myeloid cell leukemia-1(mcl-1) is an anti-apoptotic member of B cell lymphoma-2(bcl-2) genesuperfamily, which plays an important role in the regulation of apoptosis. Because mcl-1directlyregulates cell differentiation and cell cycle, it is closely related with the occurrence and development oftumor. ChanSu is made of dorsal secretions of several Bufo species, commonly used in the prescriptionsof traditional Chinese medicine for treating many diseases including cancer. In current study, mcl-1cDNA was obtained firstly during screening of the skin plasmid cDNA library of Japanese toad Bufojaponicus formosus by colony polymerase chain reaction (PCR), whose sequence was analyzed bybioinformatic methods, meanwhile the homology analysis, the prediction of the structure and functionof its encoding protein were done. Secondly, to clarify if mcl-1is expressed in Chinese toad B.gargarizans, the PCR was performed with its dorsal skin first strand cDNA as the template and a pair ofspecific primers, which were designed based on the mcl-1cDNA sequence of B. japonicus formosus.Thirdly, to obtain the recombinant protein of MCL-1, the recombinant plasmids for prokaryoticexpression of two Bufo species were constructed, and the recombinant proteins were induced, whichwere purified and used as antigen to prepare mouse anti-MCL-1antiserum. This study will become thebasis for the further studies on MCL-1functions and its related drug discovery.The main results are as follows:1. The sequence of Bufo japonicus formosus mcl-1was obtained (GenBank accession number:JX219482), whose full length cDNA was1090bp with an open reading frame (ORF) of639bpencoding212amino acid residues. MCL-1homologous analysis indicated high similarity with humanMCL-1including conserved motif, functional domain, membrane spanning domain and multiplephosphorylation sites, which suggests the value to further study of Bufo MCL-1.2. Thirteen mcl-1cDNA clones were obtained form the skin first strand cDNA of B. gargarizans.This will become the basis for the MCL-1sequence analysis of Chinese toad by bioinformaticsmethods.3. The prokaryotic expression construct of pET-28b-mcl-1was made and the expression of the recombinant protein of MCL-1was confirmed by SDS-PAGE and western blot. This study will becomethe basis for purifying the recombinant protein and preparation of its antiserum, and the further studieson the biological functions of MCL-1.4. Anti-mouse antiserum of two kinds of Bufo MCL-1showed excellent specificity, which lays afoundation for detecting of MCL-1protein expression in Bufo organism and its related anticancer drugdevelopment.