Preliminary Studies On Recombinant Expression Of Three Antimicrobial Peptides From Fenneropenaeus Chinensis And Their Application

Now the problem of aquacultural disease is still one of the importantissues that limit the development of the aquaculture. Antibiotics or chemical drugs areused to prevent or treat the diseases. Drug abuse affects the quality of aquaticproducts seriously, leading to food safety problem and the pollution of environment.Ultimately it threatens to human health. Antimicrobial peptides (AMPs), withnon-specific inhibition, have potential application value as new antimicrobial drugs,causing no drug resistance of the pathogen. In this paper, three kinds of antimicrobialpeptides from Fenneropenaeus chinensis were studied for their preliminaryapplications in aquaculture to supply a reference for antimicrobial peptidesapplications in the future.Two C-terminal coding sequences of Fenneropenaeus chinensis hemocyaninFcHC were cloned into the Pichia pastoris expression vector pPIC9K to produce thepPIC9K/FcHC-C yeast expression vectors. The constructed vectorspPIC9K/FcHC-C1and pPIC9K/FcHC-C2were linearized by Sal I enzyme, andtransformed into Pichia pastoris GS115using PEG-mediated transformation method.PCR identified transformants were screened by G418selected YPD plates. The P.pastorist transfomants of pPIC9K/FcHC-C expressed the two AMPs by methanolinduction. The results of Tricine-SDS-PAGE and Western blotting indicated thatrecombinant FcHC-C1and FcHC-C2peptides (rFcHC-C1and rFcHC-C2) wereexpressed successfully. An inhibition assay showed that rFcHC-C1and rFcHC-C2asanionic AMPs had antifungal and antibacterial activities. The application of recombinant antimicrobial peptide penaeidin3-2(rPen3-2) inaquaculture and its function mechanism were studied. rPen3-2was produced by using20L fermentor. After methanol induction for70hours, the recombinant peptideconcentration reached maximum. Fermentation supernatant was processed viadifferent protein purification methods. The results showed that target protein couldn’tbe obtained using hydrophobic chromatography and obtained a little using the ionexchange chromatography, however the recombinant antimicrobial peptides could begathered by nickel column affinity chromatography with high purity. The effect ofrPen3-2at different times and different concentrations was studied, and the resultsshowed that rPen3-2with higher concentrations had significant antibacterial activity,but it was also related to the growth of the microorganisms in the environment.Maybe the antimicrobial ability of rPen3-2relied on limiting the synthesis of largemolecular weight proteins in bacterial. The results analyzing of interaction betweenrPen3-2and antibiotics showed that rPen3-2and penicillin were indifference inAeromonas hydrophila. However, an antagonistic relationship of antibacterial activityexisted between rPen3-2and penicillin when using them in Micrococcus luteus. Theeffect of flora in aquaculture water was not significant compared with control whenadding30mg/L and90mg/L rPen3-2respectively.In summary, both penaeidin3-2(Pen3-2), as cationic antimicrobial peptides, andhemocyanin C-terminal fragment (FcHC-C1and FcHC-C2), as anionic antimicrobialpeptides, have good antibacterial activities. Recombinant protein rFcHC-C1andrFcHC-C2has the potential for disease control of animals and plants. Thefermentation process is conducive to get a large number of antimicrobial peptides forapplied research. The use of antimicrobial peptides rPen3-2has to pay attention to thetiming and dose. The effect of rPen3-2with low concentration is not significant whenthe bacteria thrived. This work provided a reference of scale production andapplication of antimicrobial peptides in aquaculture.