Co-transfection Of An Aspergillus Fumigatus Chitosanase Gene With A Reporter Gene

According to the published DNA sequence of Aspergillus fumigatus chitosanase(Csn) gene, eight long single DNA strands ,about 100 nucleotides each, and four DNA primers were designed and synthesized. By PCR, eight DNA single strands were connected into a complete chitosanase sequence of 644 bp. This chitosanase sequence was not identical to its wild type, and some point mutations were introduced into its DNA sequence by special design of those eight DNA strands. These mutaions did not change amino acid composition of the chitosanase, however, the codons were changed into mammalian cells favorites, usually mouse. The Csn gene was cloned into plasmid pGEM?-T easy and verified by DNA sequence analysis. Thereafter, the Csn gene was subcloned into the fusion-protein expression vector pGEX-3X and the eukaryotic expression vector pTracer-Csn.Then the recombinant plasmid pGEX-3X-Csn was transformed into E.coliDH5 α and the transformant was induced for expressing with 0.5mM IPTG.. The expressed product was analyzed by SDS-PAGE, and the fusion protein GST-Csn was purified by affinity chromatography. By factor Xa digestion GST-Csn was cleaved and GST was taken out by another chromatography. The bioactivity of the recombinant chitosanase(rCsn) was also analyzed, as a result the recombinant Csn had a strong ability of degrading chitosan, which was much higher than that of lysozyme.In the gene transfection experiment in vitro,the cell line C₂C₁₂ was used as host cell and plasmid pTracer-LacZ containing LacZ reporter gene was used as target DNA.Then pTracer-Csn and pTracer-LacZ were co-transfected with lipofectamine...