Fusion Expression And Functional Research Of R-PA/SP-B Based On ALI

Surfactant protein B(SP-B)was from Alveolar Type Ⅱ Epithelial Cells(ATII),which constituted the hydrophobic protein of pulmonary surfactant(PS).Its main function was promoting phospholipid molecular diffusion and increasing monolayers stability,improving the lipid-protein complex surface active and alveolar gas-liquid interfacial surface tension.Acute Lung Injury(ALI)of inflammatory factor will lead to Alveolar Type Ⅱ Epithelial Cell morphology and function changed with surfactant synthesis reduced.SP-B synthetic reduced,activity decreased and component changed could further aggravate the pathological process of ALI.Acute Pulmonary Embolism is an early relative mark of Acute Lung Injury,recombinant reteplase(r-PA)was the third generation of thrombolytic drugs,which can be effective treatment of heart,lung infarction.The fusion protein of r-PA and SP-B can develop a new gene engineering drug for ALI replacement therapy.Objective:The study was based on the construction and expression of fusion protein of r-PA and SP-B,through exploring the characteristics of the fusion expression of SP-B,providing a characteristics could not only play a reteplase thrombolytic activity,and the SP-B could be used with membrane binding ability of cell membrane localization in Alveolar Type Ⅱ Epithelial Cells,and developing a new type of genetic engineering drugs for ALI replacement therapy.Methods:(1)Study on the characteristics of SP-B fusion expression.SP-B could not be obtained by using the conventional synthesis method of amino acids.Firstly,through the synthesis of mature SP-B cDNA and plasmid pGEX4T-1 with GST-tag and SP-B will be constructed.After induced with IPTG,Western Blot was used to detect the expression of SP-B and purification of protein GST/SP-B.(2)Construction of r-PA/SP-B fusion expression vector.According to the characteristics of the fusion expression of SP-B and synthetic r-PA gene,r-PA/SP-B expression gene,constructed to extract plasmid in eukaryotic vector pEZ-M03 enzyme digestion and gene sequencing,and then transfected into CHO-K1 cells,cells expressing product was detected by Western Blot.(3)Fluorescence localization of r-PA/SP-B cells.After culture CCL149 cells,theeffects of cationic liposome transfection and transfection efficiency,the fusion expression vector by transfection method into the CCL149 cells,using vector carrying eGFP and with DAPI dye staining and Di Ⅰ cell membrane,cell fluorescence localization of r-PA and r-PA/SP-B,clear of SP-B function.(4)The study of ALI based on r-PA/SP-B.Construction of acute lung injury cell model,using a cell Raman spectroscopic technique and analysis plasmid was transfected into CCL149 cells r-PA/SP-B fusion protein and cell membrane phospholipids structure interaction and determination of fusion protein in r-PA fibrinolytic activity by fibrin agarose plate method.Result:(1)By lowing temperature and prolonging time method and GST-tag H2 N end can fusion with the-COOH end of SP-B expression form of soluble protein,which could help promoting dissolution of recombinant SP-B in the supernatant,hydrophobic SP-B and hydrophilicity could be cleared,molecular weight larger label in the host to form fusion protein.(2)Using genetic engineering techniques were used to construct the r-PA expression vector pEZ-rPA and r-PA/SP-B fusion expression vector pEZ-rPA-SPB and the-COOH terminal carrying e GFP gene,analysised by enzyme digestion and gene sequencing method,and proving the vector was successfully constructed.By SDS-PAGE and Western Blot detection,the expression product could be positively reacted with r-PA antibody and SP-B antibody,and the product was r-PA protein and r-PA/SP-B fusion protein.(3)Green fluorescent signal in the CCL149 cells mainly in the cytoplasm,the distribution of r-PA/SP-B green fluorescence signal in the cell mainly concentrated on the cell membrane that r-PA/SP-B fusion protein specific expression in CCL149 cells,and fusion protein SP-B could function independently,its position and function in the cell membrane.(4)Screened suitable for using in the preparation of cell injury model of LPS optimal dose with final concentration of 10 ug / L.In LPS induced cell injury model,r-PA and r-PA/SP-B fusion protein in cells in the normal expression and function was not affected by LPS,and membrane binding ability of SP-B did not lose.(5)Through the r-PA/SP-B in alveolar type II epithelial cells overexpression could be used with SP-B membrane-bound characteristics,maintain cell membrane phospholipid bilayer structure layer stability.Reducding damage could be caused by LPS and cytokinesof Alveolar Type Ⅱ Epithelial Cells.