Expression, Identification Of Bioactive Characterization And Antibacterial Kinetics Of Brucellaphage Tbilisi Lysin Gene

Brucellosis, a disease caused by Brucella, which are Gram-negative coccobaccilli that are nonspore-forming, nonmotile, and nonencapsulated, continues to be a worldwide zoonotic disease. The world more than 170 countries and area reported annually to the WHO made 500,000 cases. In the past fifteen years, along with the economic policy development, and each region paid different attention, the quarantine area had changed, so that the developed country also has been in epidemiologic area. Brucellosis in China spreaded to 28 provinces and municipalities, and rebound and resurgence for the disease epidemic in recent years has brought enormous economic impact of animal husbandry, and threaten seriously to the human health. At the same time, brucella is also considered to be one of the most important biological warfare agents and bioterrorism agents. As a result of its unique biological features and the damage effect, as early as namely are listed B kind of standard biological agent as in the 1950s by the US military, which has more possible been used by the enemy opposite party from the strategy and the tactic. This presented the challenge to our country and our army on biological protective capability. At present, (a) safety and effectiveness of veterinary vaccine for brucellosis have many limitation, and there is so far not a safe and effective products for human vaccine; (b) brucellosis has a long time to clinical treat and more serious resistance appeared; (c) chemistry disinfectant has high stimulation and corrosion to the equipment and causes the environmental pollution, the pathogen resisting force increasing, ecological equilibrium destruction and so on.Therefore based on the above three factors, developing the new generation safety, highly effective, moderately-priced anti-brucella drug has become our army and even our country's life scientific important topic.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 1920s 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, the bacteriophage lytic enzyme "lysis from without" has been effect to become the expanded bacteriophage biological application category as well as the better enhancement its antibacterial effect scientific foundation.The Brucella bacteriophage has many kinds of multi-price decomposition bacteriophage (for example Tbilisi, Weybridge, Berkeley, Firenze and so on), its lytic enzyme (lysin) may direct action many kinds of Brucella (including many kinds of R and S) the host cell wall, the effect high endophilicity be with the kind the special cell wall sugar base part related, therefore the bacterium produces with difficulty to the lytic enzyme resistance. Therefore, as a treatment, decontamination of antibacterial agents has obvious advantages, has made it possible to become a specialized damage to the bacteria, "Enzymology of disinfections."Therefore, our study first explored the biologic nature and molecular character, and predicted the phage lysin structure and analysis the function domain, cloned the full sequence of phage lysine gene, and successfully constructed its recombinational plasmid, then expressed, purified the fusion protein of phage lysin. Finally we examined its bio-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. Biologic nature and molecular characterization of Tbilisi. One of our objectives was to show the morphology with advanced electronic- microscope technology, and DNA restriction endonuclease profiles of Tb. Finally, we used RAPD (random amplified polymorphic DNA)/AP-PCR (arbitrarily primed PCR) fingerprinting analysis to indicate the difference between Tb and host strain DNA. We would hope to generate a new scientific understanding for the use of Brucellaphages. Tb was classified as a member of the Podoviridae family with icosahedral capsids (57±2 run diameter) and short tails (32±3 nm long). And Brucellaphage DNA was double stranded and unmethylated; its molecular size was 34.5 kilobase pairs. Some sequences were awareness by using RAPD analysis, TA cloning technology, and structural proteins were observed by using SDS-PAGE. Thus, the results have laid the foundation for the expanding usage of brucellaphage's basic mechanisms and practical applications.2. Bioinformatic analysis of brucella phage key enzyme of lysis function. We have predicted the 3D structure of brucellaphage Tbilisi hydrolase LzA by means of homology modeling that deals with the structure prediction based on the sequence homologies with the proteins of known structures. We have assessed the quality of LzA final model and discussed the active sites for catalytic triad, which allows us to make a hypothesis on the structure-function relationship in phage hydrolase. With homology modeling techniques, molecular mechanics, and molecular dynamics methods, a 3D structure model of LzA is created and refined. This model is further assessed by Profile-3D and ProStat, which confirm that the refined model is reliable. LzA, as the model predicts, is anα/βprotein consisting of two major (αandβ) domains, connected by a long a helix. The model further revealed that the protein contains 12 helices, 9 strands. The final model of LzA was verified through the program VERIFY 3D and more than 90% of the residues were in the favorable region. This new insight may improve our understanding of the molecular and functional organization of other, more complex hydrolases, and supports this idea to become one feasible method that anti-brucella relies on the bacteriophage hydrolytic enzyme function.3. The cloning of full sequence of Tbilisi phage lysin gene. The amplification of full sequence of Brucella phage Tbilisi phage lysin: by optimizing the conditions for PCR, we amplified the full sequence of phage lysin gene, laying the solid foundation for further producing of phage lysin fusion proteins. We inserted the full sequence of phage lysin gene into prokaryotic expressing vector pET32a(+), and succeeded in constructing the recombinant plasid of phage lysin(pET32a(+)-LzA), which was confirmed by Kpn I and Xho I double-enzyme incision and DNA sequencing, to find that its has correct sequence and reading frame.4. The expression and purification of full sequence of Tbilisi phage lysin gene. The recombinant plasid of phage lysin was transformed into BL21 (DE3) 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 90%, indicating that affinity chromatography could realize the further purification of phage lysin fusion protein. Target protein concentration examined by Bradford displayed 10mg/ml.5. The antibacterial kinetics of phage lysin fusion protein. With digging holes is to determine the bacteriophage lysin protein to Brucella's bacteriostasis activeness. The result showed that in joins in the reorganization bacteriophage Tb bacteriophage decomposition zymoprotein hole, might form the diameter approximately is the 0.5～2cm transparent ring, but in the negative comparison's hole, damped ring's formation not transparently. The preliminary proof lysin gene protein has certain anti-brucella activeness.6. Environment stability of bacteriophage lysin gene fusion protein. Observed under the different acid-base environment, different temperature and different ion concentration condition, environment stability of phage lysine has good performance and has more possibility which uses in the outside environment.To sum up, our study explored the biologic nature and molecular character, and predicted the phage lysin structure and analysis the function domain, amplified the full structure sequence of phage lysin, expressed and purified this protein to acquire its biologically active form. By examining its in vitro activity, we found that the fusion protein has anti-brucella activity, which provides the very foundation for its further study and possible clinical application to the treatment of infectious diseases.