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The Cloning,Expression And Activity Verification Of Pseudomonas Aeruginosa Phage PaP3 Polysaccharide Depolymerase-PSD

Posted on:2007-01-12Degree:MasterType:Thesis
Country:ChinaCandidate:M JiaFull Text:PDF
GTID:2120360272461260Subject:Microbiology
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The biofilm is a complex aggregation of microorganisms marked by the excretion of a protective and adhesive matrix, thereby playing a crucial role in the adhesion, planting of bacteria and its anti-phagocytic activities. Biofilms enable bacteria to resist numerous outside unfavorable factors and environment, and therefore are believed to be the source of many persistent and chronic bacterial infection. It has been found that biofilms are involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections (NIH 2002). Since the bacteria with biofilms are nearly resistant to all antibiotics, biofilms have warned the medical researchers to the point that they have threatened the very health of human species. Therefore, it is imperative that some measures should be found in order to destroy the bacterial biofilms, which is of great importance not only for the block of bacteria's pathogenic component but also for increasing the sensitivity of bacteria to antibiotic treatment and ultimate killing of resistant strains of bacteria. Furthermore, effective control of bacterial biofilms also serves useful purposes in some other fields like industry, nautical development, marine aquaculture, and so forth.Bacteriophage, phage in short, is a virus that infects bacteria leading to its lysis. Phages were tried as anti-bacterial agents after their discovery. However antibiotics, upon their discovery, proved to be more practical. Research on phage therapy was largely discontinued in the West, but phage therapy has been used since the 1940s in the former Soviet Union as an alternative to antibiotics for treating bacterial infections. The evolution of bacterial strains through natural selection that are resistant to multiple drugs has led some medical researchers to re-evaluate phages as alternatives to the use of antibiotics. Unlike antibiotics, phages adapt along with the bacteria, as they have done for millions of years, so a sustained resistance is unlikely. Additionally, when an effective phage has been found it will seek out the bacteria and continue to kill bacteria of that type until they are all gone.Therefore, phage therapy has become a promising means in dealing with antibiotics resistent strains of bacteria and has been widely applied to clinical studies in many countries. So far, there have been reports describing serious complications caused by phage therapy.A phage has to break exopolysaccharide (EPS) before its entrance into cytosol. It has been shown that phage does produce specific enzymes to degrade EPS. Although these enzymes only degrade biofilms and strip the protection layer of bacteria, they serve to greatly lower the possibility of bacterial adhesion, planting and anti-phagotic activities, and thereby facilitating the effective treatment for biofilms bacterial infections in clinical aspects.Funded by the NFSC, our lab has successfully sequenced the genome of Pseudomonas aeruginosa phage the result of which has been added to Genbank (NC 004466). We discovered PaP3p02 gene whose product is conceived to be a member of the family of polysaccharide depolymerase(PSD). This gene product can degrade EPS and is therefore considered to be the promising zymdogic disinfectant to destroy bacterial biofilms.Therefore, our study first cloned the full sequence of polysaccharide depolymerase gene, and successfully constructed its recombinational plasmid, then expressed, purified the fusion protein of polysaccharide depolymerase. Finally we examined its bacteriostasis activity in vitro so as to investigate the feasibility and effectiveness of utilizing it as a zymdogic disinfectant. The content and results of our study include the following aspects.1. The amplification of full sequence of Pseudomonas aeruginosa phage PaP3 polysaccharide depolymerase: by optimizing the conditions for PCR, we amplified the full sequence of polysaccharide depolymerase gene, laying the solid foundation for further producing of polysaccharide depolymerase fusion proteins.2. The cloning of full sequence of Pseudomonas aeruginosa phage PaP3 polysaccharide depolymerase gene. We inserted the full sequence of polysaccharide depolymerase gene into prokaryotic expressing vector pQE-31, and succeeded in constructing the recombinant plasid of polysaccharide depolymerase(pQE-PSD), which was confirmed by BamHâ… and Hindâ…¢double-enzyme incision and DNA sequencing, to find that its has correct sequence and reading frame.3. The expression and purification of full sequence of Pseudomonas aeruginosa phage PaP3 polysaccharide depolymerase gene. The recombinant plasid of polysaccharide depolymerase was transformed into JM109 in order to obtain stably expressed clone. During the processing of inclusion body, we found that the target protein is expressed to be secretory proteins, with 80% of which exist in supernatant. The amount of protein expressed in supernatant is four times larger than that expressed in inclusion body. The target protein in supernatant was purified through Ni-NTA column and filtration chromatography amounting to a single lane in SDS-PAGE without interfering lanes and a purification of more than 95%, indicating that affinity chromatography could realize the further purification of polysaccharide depolymerase fusion protein. Target protein concentration examined by BCA displayed 2.278mg/ml.4. The biological activity of polysaccharide depolymerase fusion protein. We extracted the EPS component from Pseudomonas aeruginosa biofilms and examined the degrading activity of polysaccharide depolymerasethe fusion protein. The results showed that the introduction of polysaccharide depolymerase fusion protein degraded EPS illustrated by the forming of lucid inhibition zone.5. Bacteriostasis activity of polysaccharide depolymerase fusion protein in vitro. We observed the changes in sensitivities of Pseudomonas aeruginosa PA3 to four antibiotics(gentamicin, ciprofloxacin, gatifloxacin, ceftazidime) brought by the presence of polysaccharide depolymerase by microdosis doubling dilution. We found that polysaccharide depolymerase induced the decrease in MIC,MBC of all the four antibiotics with ceftazidime being the most influenced, which indicates polysaccharide depolymerase has antibiotic antivity. Labeling the biofilms of Pseudomonas aeruginosa PA3 and treating them with polysaccharide depolymerase fusion protein, we discovered that there were less labeled green EPS and fewer amount of bacteria wrapped in EPS compared with control group. These results demonstrated that polysaccharide depolymerase can specifically degrade the EPS in biofilms and exert antibiotic effect.To sum up, our study amplified the full structure sequence of polysaccharide depolymerase, expressed and purified this protein to acquire its biologically active form. By examining its in vitro activity, we found that the fusion protein has degrading activity against EPS in biofilms, which provides the very foundation for its further study and possible clinical application to the treatment of infectious diseases.
Keywords/Search Tags:Pseudomonas aeruginosa, Biofilm, exopolysaccharide, polysaccharide depolymerase, gene cloning, Pqe-PSD, prokaryotic expression, protein purification, fusion protein, bacteriostasis activity
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