Research On Localization Of SP-B And Biological Activity Of Mimic Peptide

Hydrophobic pulmonary surfactant protein B (Surfactant protein B, SP-B) is theearliest discovered lung surfactant protein.As a result of interacting with the phospholipids,they formed a stable pulmonary surfactant (pulmonary surfactant PS) molecular layer inalveolar surface to maintain the morphology of alveolar and good lung compliance.Pulmonary surfactant has became one of the important drug for the treatment of ALI(Acute Lung Injury, ALI) in recent years.Currently, the vast majority of pulmonarysurfactant drug listed are natural extracts. The main component of the mixture is lungsurfactant protein and phospholipids and SP-B is one of the ingredients of the mixture thatplayed the important functional role.But the limitations of material sources, highproduction costs,the immune response and bacterial infections may occu in the treatmentand other factors had limited to the clinical application.Synthesis the key ingredient ofpulmonary surfactant is the research trend in recently years.Objective:In this study, we design and screen a optimum SP-B mimic peptide structure base onthe in-depth understanding of localization of SP-B in rat lung and the charge distribution,secondary structure, hydrophobic characteristics of the SP-B sequence.Then, Synthesis themimetic peptide.Vitro activity, influence on the conformation and function of the lipidstructure and pulmonary compliance of rat isolated lung are measured to lay a foundationfor further researching and developing artificial lung surfactant.Methods:①Localization of SP-B protein: Detect the localization of SP-B protein in lung tissue byimmunofluorescence.Constructed recombinant plasmid pEGFP-N2-SP-B, the plasmidwas transfected into CCl-149and A549cell lines by liposome.②Design, screening and synthesis of mimetic peptides: Design and screen of mimeticpeptides by bioinformatics tools and synthesis by standard Fmoc solid phase chemicalsynthesis method.③Vitro activity of mimic peptide: Build liposomes simulation film,Determine the activityof the mimetic peptides in vitro by Wilhelmy film balance.④Interaction of mimic peptide and lipid: Detect the trans,twisted conformation changes of lipid by Raman spectroscopy..⑤Activity research in vivo: Build rat isolated lung lavage model, Determine the isolatedlung compliance of rat.Result:①Confirmed the localization of SP-B protein in lung tissue,Plasmid pUC-T-SP-B andpEGFP-N2-SP-B are successfully constructed and sequenced correctly.The liposomaltransfection results showed that SP-B mainly located in the cytoplasm of A549cell lineand in cell membrane of CCL-149cell lines.②Designed the mimetic peptide KAAVVKAAVVKAAVVKAAVVK by bioinformaticstools.Commissioned GL Biochem to synthesis the polypeptide.HPLC resport showedthat purity is about98.6%and mass spectrometry result showed molecular weightresults consistent with the theoretical value.③Successfully built the biomimetic membranes by phospholipid.The preliminary activityin vitro was measured by wilhelmy film balance.The result showed that the peptidecould effectively reduce the tension of gas-liquid surface.④Raman spectroscopy analysis results showed that the ordered conformation of C-Cskeleton had reduced and the chain disorder conformation enhanced after mixed thepolypeptide with DPPC and DPPG.C-H stretching vibration results showed the fluidityof the bilayer membrane enhanced after added the polypeptides.From the results wecould conclude that the polypeptide played a role like SP-B in PS.⑤Vitro static lung compliance measurement results showed that the mimic peptidepreparation could significantly improve the lung compliance.