Research Of Recombinant Expression And Antibacterial Functional Of Precursor Surfactant Protein B

Precursor surfactant protein B(pro SP-B)is a precursor protein in the formation of pulmonary surfactant protein B,which plays an important role in the formation and regulation of pulmonary surfactant protein B.The hydrophobicity of the pro SP-B amino acid sequence and the uniqueness of its structure were classified as a member of the saposin-like proteins(SAPLIPs)family by the scholar.The SAPLIPs family members as a family of proteins which with unique structures and broad-spectrum biological functions,the antimicrobial activity is one of its most important functions.In recent years,with the abuse and irrational use of antibiotics in the field of food and medicine,causing drug resistance,pandrug-resistant,cross resistant,and even the emergence of superbacteria,it is lead to antiinfection problems become more and more grim,human health and life safety facing great threat.Therefore,development and research of new high efficiency,low toxicity,with new antibacterial mechanism of the drug is imminent.With the increase of antibiotic side effects and resistance,antimicrobial peptides which with broad-spectrum antibacterial activity,produced by the physiological activities of the organisms,have becoming a new class of antibiotics which are used to replace traditional antibiotics.In this study,we constructed the prokaryotic expression vector p GEX4T-1-pro SP-B by gene engineering technology.The pro SP-B protein was successfully expressed by IPTG,and the protein was successfully purified by GSTrap FF 16/10.Then we used CCL149 cells as the experimental object to analyze the localization of pro SP-B protein in cells and the toxic effect of pro SP-B protein,which laid a foundation for the antibacterial effect and mechanism of pro SP-B protein.Then,the antibacterial activity of prosp-B protein was investigated by using Staphylococcus aureus,Escherichia coli,Candida albicans,Trichophyton rubrum,Gypsum-like microsporidia,Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa as the research object.Staphylococcus aureus as the key research object,to study the mechanism of antibacterial protein,for the development of a new type to lay the foundation.Objective: In this study,the aim of this study was to construct,express and purify the recombinant protein of pro SP-B by genetic engineering technology,analyze its potential toxicity, antibacterial activity and antibacterial mechanism,lay the experimental foundation for prosp-B development as a new type of potential antimicrobial agent.Methods:(1)Construction p GEX4T-1-pro SP-B prokaryotic expression vector,analysis pro SPB fusion expression characteristics.The amino acid sequence of pro SP-B was highly hydrophobic and was harder to be obtained by chemical synthesis.The target gene pro SP-B was constructed on the p GEX4T-1 plasmid by genetic engineering.The expression of pro SPB was induced by IPTG,detected the expression of pro SP-B protein by SDS-PAGE and Western Blot,and purified obtain the pro SP-B protein.(2)Study on cell fluorescence localization and cytotoxicity of pro SP-B protein.The eukaryotic expression vector p EGFP-N2-pro SP-B was constructed and transfected into CCL149 cells,and using GFP gene carried on the vector,stained with DAPI and stained with Di I cells to analyze the localization of pro SP-B in cells.Then,the cytotoxicity of pro SP-B protein was examined by co-incubation of CCL149 cells with pro SP-B protein.(3)Study on antimicrobial activity of pro SP-B protein.Staphylococcus aureus,Escherichia coli,Candida albicans,Trichophyton rubrum,Gypsum-like microsporidia,Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were cultured and the antimicrobial activity of pro SP-B protein was investigated by filter paper diffusion method and antibacterial spectrum.(4)Study on antibacterial mechanism of pro SP-B protein.The use of Staphylococcus aureus as the purpose of bacteria,analysis of pro SP-B protein antibacterial mechanism by using propidium iodide(PI)and lactate dehydrogenase(LDH)activity assay.Results:(1)The prokaryotic expression vector of pro SP-B was successfully constructed and the soluble expression of recombinant pro SP-B protein was successfully promoted at a lower temperature and prolonged time.The pro SP-B protein was successfully purified and the protein content was 1 mg/m L by BCA.(2)The GFP-pro SP-B fusion expression vector p EGFP-N2-pro SPB is successful construct by molecular biology technology.The results showed that the distribution of GFPpro SP-B green fluorescent protein in CCL149 cells was mainly concentrated on the cell membrane,while the green fluorescence of GFP was distributed in the cytoplasm of CCL149 cells,indicating that the pro SP-B fusion protein have specific expression in CCL149 cells,and the pro SP-B protein can play its membrane localization role and function.(3)The cytotoxicity of pro SP-B protein was determined by co-culture of protein and cell,and the IC50 was about 1595 μl/m L,the cytotoxicity of pro SP-B protein was determined by cytotoxicity grading standardwas micro toxicity.(4)The sensitivity of the protein to Staphylococcus aureus,Escherichia coli,Candida albicans,Trichophyton mentagrophytes,Gypsum-like microsporidia,Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa were analyzed by filter paper method,and calculate its MIC.It was found that the pro SP-B protein had some inhibitory effects on the bacteria and fungi,and the effect on the Gram-positive bacteria was greater than that the Gram-negative bacteria,and the effect on the bacteria was greater than that fungi.(5)A single choice of Staphylococcus aureus as an experimental object,study the primary antimicrobial mechanism of pro SP-B protein was determined by PI fluorescence staining and LDH extravasation.And binding of pro SP-B to fluorescent cells localization,our preliminary found the antibacterial activity of pro SP-B protein is the role of protein in the cell membrane of bacteria by changing the bacterial membrane structure and affect the bacterial cell membrane protection and regulation,thereby inhibiting or killing bacteria.