Study On The Recombined Cationic Anti-Microbial Peptide

The target gene with length of 207bp was designed and synthesized for our research. The gene physical map was drawn after the target gene was inserted into Vector pBluescript-M13. It verified the correct reading fragment by DNA sequencing. Secondly, we selected yeast Pichia pastoris to be the gene-expressing host and the secreting plasmid pPIC9 was as vector to construct recombined plasmid pPIC-Atmp. The recombined plasmid pPIC-Atmp was electro-transporation into host yeast Pichia pastoris GS115(his-Mut+) after the liberalization by restriction endonuclease BglⅡ. By comparison of the growth on MM and MD media plate, the his+Muts phenotype transformant had been picked up for further screen. The purpose protein cationic antimicrobial peptide was expressed through fermenting transgenic strain JCC-2 in a small scale and the fermentation supernatant had been analyzed through Tricine-SDS-PAGE. It proved the correct expression of recombined cationic anti-microbial peptide and the peptide was in the form of polymer in the fermentation supernatant according to the molecular weight of expressed purpose peptide. The monomer was attained through another Tricine-SDS-PAGE after the polymer treated withβ-mercaptoethanol and the molecular weight of monomer demonstrated to be about 6000 Dalton through the analysis with Gel analysis system. The max expressed quantity of the recombined peptide is up to 29.67mg/ml. The anti-microbial experiments in vitro proved that the recombinant peptide (the fermentation supernatant about 30ul, 96h) could inhibite the growth of Sarcina subflava Ravenel, Escherichia coli and the inhibited diameters were 2.6cm, 2.36cm, respectably. The anti-microbial diameter of fermentation supernatant of different time growed with the expression time went on and the diameter of fermentation supernatant to Sarcina subflava Ravenel was bigger than to Escherichia coli. Through optimization, the part optimized fermentation parameters had been attained. The experiment results showed that the fitful growing medium of JCC-2 is BMGY and the starting pH of BMGY is 6.0. The max biomass, which reached 24.7g/l, could be attained after 70 hours cultivating on the BMGY. The expressed quantity of purpose peptide would be up to 29.67mg/ml when the strains was cultivated on the inducing medium BMMY 96 hours. In short, the recombined plasmid pPIC-Atmp had been linearized before it electro-transducted into host strain GS115 and the phenotype transformant had been screened out as well as multi-copy transformant. The target peptide had been analyzed and proved its correct expression. The anti-microbial activity in vitro had been checked and the optimized fermentation parameters had been attained in order to the further research.