INVESTIGATION ON THE IN VITRO ANTIBACTERIAL ACTIVITY OF HUMAN Α DEFENSIN 5(Α-HD-5)EUKARYOTIC EXPRESSION PRODUCT

[Objective]To explore the potency of HD-5 as an alternative to traditional antibiotics,so as to provide experimental basis for the research of HD-5 antibacterial bioactivity.[Methods]Recombinant Pichia pastoris expression vector pGAPZαA-HD5 was constructed and transformed into Pichia pastoris GS115 cells.Positive colonies were screened out on plates containing Zeocin.Recombinant strain were cultivated in a shaking air incubator for about 48 hours and then centrifuged.Supernatant liquid was obtained and freeze-dried.The freeze-dried powder was dissolved and then diluted to obtain stock solution(solution I),5-fold concentrated solution(solutionⅡ)and 10-fold concentrated solution(solutionⅢ),which all contained HD-5.Culture supernatant(0 solution)absence of HD-5 served as an negative control.Inhibition zone diameter were measured as their impact on standard strains of Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa detected by drug sensitivity test agar-well diffusion method.[Results]As HD-5 concentration gradually increased,inhibition zone diameter of 0,I,Ⅱ,Ⅲ solution against E.coli was 0mm,(16.56 ± 0.45)mm,(20.09 ± 0.67)mm,(26.72 ± 0.54)mm,which against Staphylococcus aureus was 0mm,(11.45 ± 0.13)mm,(18.09 ± 0.32)mm and(25.50 ± 0.21)mm.Only solution Ⅲ showed inhibition zone against Pseudomonas aeruginosa(17.45 ± 0.56mm).[Conclusion]In vitro experiments revealed that HD-5 eukaryotic expression product exhibited prominent inhibitory effect against Gram-positive and gram-negative bacteria,suggested its antimicrobial potential clinical applications as a source of novel antibiotics.