Construction Of Polycistronic Expression Vector Of Antifungal Peptides CGA-N46Gene And It Identification

Chromogranin A exists in various animal endocrine organizations andneuroendocrine cell one of eosinophilic chromium particle acidic glycoprotein. One ofthe N-terminal derived peptides, CGA-N46, is a short peptide with strong antifungalactivity to Candida albican and is expected to become a novel antifungal agent withlow molecular mass. In this study, in order to improve the amount of gene engineeringexpression of CGA-N46gene, a pair of restriction endonucleases to make the samestick end Nhe I and Spe I were used to construct the recombinant T vectors pT-CAN46,pT-3CAN46, pT-5CAN46and pT-8CAN46through recombining single, three, fiveand eight copies of the heterogeneous DNA fragment which contained SD sequence,start codon, CGA-N46encoding sequence and stop codon with the cloning vectorpEASY-Blunt respectively. The monocistronic, tricistronic, five-cistronic andeight-cistronic expression plasmid of CGA-N46gene pET-CAN46, pET-3CAN46,pET-5CAN46and pET-8CAN46were constructed by subcloning the CGA-N46genefragment in recombinant T vector to the expression plasmid pET-30(a). Therecombinant expression plasmids were transformed to the Escherichia coil strainRosetta to form engineered strains E. coli Rosetta(pET-CAN46), E. coliRosetta(pET-3CAN46), E. coli Rosetta(pET-5CAN46) and E. coliRosetta(pET-8CAN46). The expression of engineered Escherichia coil was studied bySDS-PAGE. The results demonstrat that all the polycistronic of CGA-N46geneexpress CGA-N46, and the expression amount of five-cistron of CGA-N46gene is thehighest, and the peptide existed in the cells mainly as soluble form after induced byIPTG. The results of this thesis provide a profoundable basis for the further researchof CGA-N46.