Study Of Molecular Modification And Function Of Porcine-?-defensin-2 In Vitro

Mammalian defensins attract wide attentions because of their strong and broad antimicrobial spectrum and pathogenic microorganisms have little resistance to them.Based on the previous research,site-directed mutagenesis and DNA shuffling were used to obtain mutants of p BD-2,the fusion protein were expressed by E.coli expression system,then explored the relationship between three dimensional structure and functions according to analysis the activities of these mutants.The main research contents were as follows:1.Site-directed mutagenesis of porcine beta defensin 2 and analysis of their activitiesBased on the previous study,we designed some mutations in order to improve the antibacterial activity of p BD-2 by increasing its numbers of positive charges;by analysis of physicochemical properties and three dimensional structures,some amino acids were selected to mutate and their engineering strains BL21-PET-mp BD2-DD1~DD6 were constructed.The result showed that the mutants showed different antibacterial activity against S.aureus and E.coli.The hemolytic test showed all the mutants have little toxicity to porcine red blood cells because of the low hemolytic ratios(-10%~10%).Mutants DD2,DD3 and DD4 were selected for salt-toleranct test,DD2 and DD3 showed good antibacterial activity in different salt concentration at different time points,which proved that mutants DD2 and DD3 had good salt tolerance.2.DNA shuffling of porcine beta defensin 2 and analysis of their activitiesDNA shuffling were carried on based on the mutated genes of porcine beta defensin 2 which were constructed in our lab,17 mutants were obtained after five cycles of DNA shuffling,six mutants DS1~DS6 were selected after physicochemical properties,three dimensional structures and the distributions of charges on the surface were forecasted.Then engineering strains BL21-PET-mp BD2-DS1~DS6 were constructed,the result showed that all the mutants had different antibacterial activity to S.aureus ATCC 25923 and E.coli ATCC 25922.Hemolytic activity assay showed that they had little toxicity to porcine red blood cell since the low hemolytic ratios of pBD-2 and its mutants(except DS2).In this study,we selected the mutants with better antibacterial activity to test their salt tolerance,the mutants DS2,DS3,DS5 and DS6 showed good antibacterial activity in different salt concentration solution,which meant the mutants also have good salt tolerance.The above results indicated that the mutants were obtained by site-directed mutagenesis and DNA shuffling,and showed different degrees of antibacterial activity however,the antibacterial activities against S.aureus and E.coli had no significant increased;Most of the mutants showed similar antibacterial activities with p BD-2,in addition,they had little toxicity to porcine red blood cells;The majority of selected mutants showed antibacterial activities in saline solution,which indicated that the mutants of p BD-2 all have good salt tolerance.The results also manifested that the antibacterial activity was not only decided by the increased net positive charge,but also connected with some other factors,such as grand average of hydropathicity,three dimensional structure and the positive charges arrangement on the molecule surface.