Preliminary Study Of Antimicrobial Mechanism Of Porcine ?-defensin 2 In Vitro

Defensins are kinds of cationic antimicrobial peptides which contain arginine and cysteine residues,and they have widespread biological activities and secreted by animals and plants.They could inhibit various kinds of pathogenic microorganisms,and didn't make bacteria produce resistance.In order to clarify the antibacterial mechanism of porcine ? defensin 2,E.coli or S.aureus after treated with p BD-2 for a certain time were identified by scanning electron microscopy,immunofluorescence microscopy and immuno-electron microscopy,as well as location of p BD-2.Details are as follows:1.In order to test the expression of p BD-2 in tissues,the p BD-2 antibodies were used by immunohistochemistry.The results showed that the polyclonal antibody could specifically recognize p BD-2 protein in kidney and intestine.2.Based on engineering strain BL21 p ET30a-p BD-2 in our laboratory,which was induced and the p BD-2 was purified.The results showed that p BD-2 could inhibit the growth of E.coli and S.aureus(109 CFU/ml),there were significant difference among doses or between times.3.1 × 109 CFU/ml of E.coli or S.aureus was co-cultured with different concentrations of p BD-2(0?g/ml,37.5?g/ml,75?g/ml,150?g/ml),after centrifugation,bacteria were fixation,dehydration,washed,froze drying and observed by scanning electron microscope.The results showed that E.coli and S.aureus were damaged differently by p BD-2,the contents were leakaged.4.On the basis of above experiment,bacteria were centrifugated after treated with different concentrations of p BD-2,and washed by PBS,then fixed,coated on a slide and carried out by immunofluorescence tests.The results showed that the p BD-2 generally distributed on the outer membrane,and perhaps inside the bacteria.So p BD-2 effected on the membrane of E.coli or S.aureus to kill bacteria,and didn't exclud that p BD-2 could enter into the intracellular to kill bacteria.5.E.coli or S.aureus treated with 150?g/ml p BD-2 for 4 h,and observed by immune electron microscopy.The results showed that p BD-2 mainly appeared in the cytoplasm of E.coli,in contrast,p BD-2 focused on the membranes of S.aureus,which meant that it was different for p BD2 to kill E.coli and S.aureus.it perhaps meant that p BD-2 killed E.coli by reacting with substance in the cytoplasm,and killed S.aureus by reacting with membrane.totally,E.coli or S.aureus was incubated with p BD-2 at different time,then observed by scanning electron microscope,immunofluorescence and immune electron microscopy.The resulted showed that it was different that mechanism of p BD-2 killing E.coli and S.aureus.which were basis for the further study and for its application in pig disease.