Construction Of Secretion System For Cationic Antimicrobial Peptide G13

In order to study the relationships between structure and function of cationic antimicrobial peptides G13, and explore effective ways of expression, we constructed a prokaryotic secretory expression system. Plasmid pET-22b (+) can deliver heterologous protein into the periplasm of E. coli cells, this mode is beneficial to purify protein. G13 and fusion protein gene were spliced together, recombinant plasmid pET-22b (+)-DPG13, pET-22b (+)-D3PG13, pET-22b (+)-C-DPG13, pET-22b (+)-C-D6PG13DP, and than induced secretion expression of G13. Results showed that only pET-22b (+)-C-DPG13 get the target protein. Although the yield below standard, but it proved prokaryotic secretory expression system is feasible for expression of G13. We need more experiments to modify the sequence of G13 and optimize the Culture Conditions.In order to evaluate the feasibility of G13 as feed additive, this experiment constructs eukaryotic secrete expression system. We designed G13 gene sequences according to codon prefernece type of yeast, and get G13 by polymerase chain reaction(PCR). After that G13 was TA cloned and sequenced. After double digests, G13 was ligated with pPIC9.we constructed the plasmid pPIC9-G13 successfully. Linearized plasmid was precipitated with ethanol treatment, than transformed into Pichia pastoris GS115 competent cells, selected His+Muts transformants, induced with the corresponding medium. Antibacterial experiment fails to detect biological activity, Tricine-SDS-PAGE didn't detect target protein. Follow-up testing should proceed to verify whether mRNA transcribed and translated, identify the reasons that why G13 can't expressed. Meanwhile, my experiments gained antibacterial peptides Pleurocidin derived from fish and human defensin peptide Pleurocidin hBD-3 gene with spliced overlap extension PCR. The recombinant plasmid pPIC9-Ple, pPIC9-HBD-3 were constructed. This two kinds of antibacterial peptides have potential application value in food and medical care.The experiment also isolated a Microcystis strain from Chaohu Lake, named chaohu-1, and its sequence has been submitted to GenBank. We use molecular biological methods identified the species is Microcystis aeruginosa. The microcystin was detected by HPLC, confirmed this algae strain produce MC - LR cyanobacteria toxin. Besides, we established a feasible process for its cryopreservation and recovery.