The Purification And Activity Assay Of The Antibacterial Peptide Isolated From The Dermal Gland Of Xenopus Laevis

In nature,the amphibians usually secrets the special substrates from skin to respond to the environmental stimulation.The secretion contains a lot of natural antibacterial peptide with small molecular weight.Due to the wide spread distribution and great number of amphibians,it will be the good resource of antibacterial peptide.In this study,we used the electronic stimulation to make the Xenopus laevis to secret the mixture and then applied HPLC to purify the mixture to get the single gradient antibacterial peptide for further functional characterization.To get the pure antibacterial peptide,the secretion was first centrifuged with ultra-high speed to get rid of big mass peptides,and the supernatant was then applied to preparative RP-HPLC with C18-300 column to obtain the single peak fractionation.The single peak fractionation was further purified with C18-300 RP-HPLC and obtained 3 antibacterial peptides.The sequence composition of peptides was checked by Edman degradation and MALDI-TOF/TOF-MS.Two newly identified antibacterial peptides were never reported and named as P1 and P2,respectively.To further characterize these two newly identified antibacterial peptides,they were artificially synthesized and checked their ability of antibacterial,hemolytic activity and anti-cancer.The results demonstrated that these two peptides not only suppress the growth of antibiotics resistant bacteria,but also show very strong inhibitory activity for breast cancer cell line,MCF-7.In the high concentration,the antitumor activity can reach 90 %,42 % and 45 % can still be yielded even at the concentration of 5 ?g/ml.The hemolytic test showed that these peptides almost don't have hemolytic activity,only minor hemolytic activity can be observed under the extreme high concentration.To investigate the potential mechanism of antibacterial activity of these peptides,the TEM was applied to check the surface change of bacterial after treated with/without these peptides.To explore the potentiality of commercial application,the mixture of peptides and adjuvant was emulsified to get the nanoemulsion.The Staphylococcus aureus were incubated with purified peptides or nanoemulsion to evaluate the minimum inhibitory concentration(MIC)at different time points.The result showed that,without emulsion,the purified peptides lose their antibacterial ability(MIC 320 ?g/ml)after 4h whereas the nanoemulsion lose the antibacterial activity till 48 h.It implies that the emulsion can extend the effect of these peptides.Taken together,our results revealed that the peptides purified from the skin secretion of Xenopus laevis can effectively inhibit the growth of bacterial with antibiotic resistance,and they also show activity of anti-caner.These results imply that these peptides might be a potential substrate for antibacterial and cancer treatment drug development.