| Brucella is a gram-negative bacterium can infect people and animals,Brucellosis were caused by the bacterium can cause human and animal infections,It is widely popular in China and around the world, to the affected areas of human health and livestock production have caused serious influence. Brucellosis by damaged mucous membranes, respiratory and digestive tract infections,it is clinically characterized by fever, abortion, infertility, chronic arthritis and nerve damage and other symptoms. Brucella is parasitic bacteria,Its pathogenesis and immune mechanism is not very clear, this is also one of the reasons is difficult to control.The purpose of the study is the foundation for the development of molecular markers of brucella ghost vaccine based on building UGPase deficient strains,producting ghost vaccine strains and studying the immunogenicity.In a previous comparative proteomics study we found that UGPase is high-expression in virulent strain and low-expression in attenuated strain. After confirm the protein is UTP-glucose- 1-phosphateuridyly–ltransferase, UGPase, one of the enzymes associated with glycogen synthesis.Bacterial ghosts is a bacteria shell which lack of bacterial cell contents(cytoplasm, nucleic acids, etc.)and without the ability to reproduce.. In natural environment bacterial ghosts comes from bacteria E. coli infected with phage PhiX174 PhiX174 after cell lysis and formation.Manmade bacterial ghosts using genetic engineering techniques, makes PhiX174 phage lysis gene E expression in target cells,In the cell surface transmembrane pore diameter 40-200 nm, resulting the changes in osmotic pressure of inside and outside the cell, the contents of the outflow formed shell morphology.In the process of forming the bacterial ghosts, the bacteria does not undergo physical and chemical denaturation process, thereby preserving the natural bacterial cell membrane structure and surface antigen,the immunogenicity is retained, as a new candidate vaccine is currently widely used in the study of a variety of gram-negative bacteria were.In this study, it constructed a suicide plasmid pBK-S-F-R containing UGP gene, the new plasmid exchange with allele by suicide plasmid-mediated, we used a two-step method to screen the recombinant strains, and the resule of PCR showed that the strain without ΔUGPase was successfully constructed.Then we used Brucella 16 M strains, AGPase deletion strains, M5 vaccine strain take a adhered test to murine macrophage RAW264.7, and animal attack drug test,The results showed that ΔUGPase deficient strains in the virulent strain 16 M Adherence to the invasion and virulence host cells are not significantly different.In order to investigate whether the strain deletion AGPase can be a possible vaccine candidate,This study contains a wide host temperature pyrolysis function expression plasmid pBBR1MCS-2-TLC into ΔUGPase deficient strains by shock, then by inducing, screening, we prepared the sheep Brucella bacteria ΔUGPase shell vaccine strain.Next, we take a experimental analysis of lytic kinetics of Brucella vaccine strain bacteria shell, optimization by inducing conditions in the culture to OD600 value of 0.5 for induction, after induction 48 h, it can be prepared by cleavage of 99.99% of the dogs bacterial ghost, the ghosts bacteriostatic number is about 5.62 × 109 cfu / mL.Last, by comparison of ΔUGPase ghost vaccine,16 M formaldehyde inactivated vaccine and M5 attenuated vaccine in the dynamics of antibodies and splenic T lymphocyte subsets analysis,ΔUGPase ghost vaccine confirmed the potential value as a marker candidate vaccine strain melit... |
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