Genetic Polymorphism Analysis Of ?-defensins From Reptile And Functional Verification Of Eukaryotic Expressed Molecules

Defensins are a group of host defense peptides that widely distributed in vertebrates,invertebrates,insect,even in fungi and plants.Besides their direct antimicrobial function,defensins also have immune-modulating activity.Defensins play important roles in host innate immune responses.Nowadays,PMX-30063,a member of defensins,has completed a phase I clinical trial for systemic infection and phase II clinical trial for acute bacterial skin infections.Thus defensin has become one of the few classes of peptides that have entered clinical studies.Peptides from reptiles have become an important source for the discovery of leading compounds,due to their strong activity and high potency.However,there are few reports related to defensins in reptiles,and they are always confined to gene cloning and lacking relative functional verification.In this study,26 novel ?-defensin peptides have been identified in NCBI database,which belong to reptilian testudinate Pelodiscus sinensis,were designated as Ps-BD1~26.Ps-BDs share some common characteristics with ?-defensin family: the precursor peptide consist of a signal peptide sequence,a short intervening region and a mature peptide.Despite high variation in mature region,it still has six conserved cysteines and GXC motif.Phylogenetic study confirmed that ?-defensins are fast evolving genes with high rates of sequence substitutions.Remarkable diversity in the mature peptide domain is a consequence of such gene duplication followed by subsequent rapid divergence.The expression profile and the three-dimension structure of Ps-BDs were studied to explore their potential defense response and structural basis for biological function.The expression levels of Ps-BD2,Ps-BD6,Ps-BD11,Ps-BD16 and Ps-BD21 in different tissues were examined by RT-PCR,which showed high expression levels of the selected Ps-BDs genes in tissues directly contact with the environment(skin,lung and stomach)or related to immune system(liver,kidney and intestine).Homology modelling showed Ps-BDs mainly adopt ?-strands and/or amphiphilic ?-helix conformations,which provided the structural basis for their biological functions.Ultimately,we used eukaryotic expression vector to express Ps-BD2 for the first time and obtained recombinant protein(rPs-BD2)successfully.Then the biological activities of rPs-BD2 were detected.The rPs-BD2 showed relatively broad-spectrum antimicrobial activity against all tested microorganisms,and was more effective to Gram-positive bacteria than Gram-negative and fungus.Meanwhile,the rPs-BD2 displayed a negligible hemolytic and cytotoxic activity to normal cells and mammals erythrocytes.It is further proved that defensins play an important role in protecting the host from pathogen invasion and the rPs-BD2 has certain guiding significance in clinical application.