The Biological Activity Of Human Beta Defensins And Their Analogs

Object: Chose Human beta-defensin1-4(hBD-1-4) as template, to design new defensin analogs. Aimed atimproving their antibacterial activity, while reducing salt sensitivity and toxicity of eukaryotic cells, andmade a speculation of structure-activity relationships and mechanism of defensins. Attempt the way to getprotein from prokaryotic cell protein expression method.Methods:(1) Defensins were synthesized by Fmoc method, and analysed their purity and structureprimarily.(2) Analysed defensins’ antibacterial activity and salt sensitivity, and observed their role ofkilling bacteria by transmission electron microscopy.(3) Mensurated the cytotoxicity (MTT method) andhymolysis of beta-defensins and its analogs.(4) Construct a expression vector to express hBD-3.Results:(1) HBD-3has the strongest antibacterial activity and salt resistant ability among hBD-1-4, itsLD90s to the bacteria in this experiment were lower than15μg/ml. At the same time, the LD90s of hBD-1were higher than100μg/ml. The antimicrobial activity of human hBD-2, hBD-4were weaker than hBD-3(P＜0.01).(2) The three N-terminal amino acid analog was more salt resistant than hBD-3while itscytotoxicity became stronger obviously, while the antimicrobacterial activity of the nine N-terminal aminoacid analog was weaker than hBD-3.(3) The I region changed analogs analogs had a stronger antibacterialactivity and salt resistant ability than hBD-3, but these ability of their N-terminal deletion analogs becameweaker.(4) We found that the analogs which had a higher salt resistant ability were always more toxic.(5)Transmission electron microscopy showed, defensin destoryed cell wall and cell membrane, and then leada death of bacteria.(6) We sucessfully expressed a fusion protein GST-hBD-3.Conclusion:(1) It is a short cut that using hBD-3as a template in defensin reconstruction.(2) TheN-terminal amino acid is important for salt sensitivity.(3) The I region of hBD-3is important for itsantibacterial activity. In later work, we will study the relationship between antibacterial activity andcytotoxicity of defensin.