| Bordetella bronchiseptica a gram negative bacterium,which can infect respiratory tract of mammalian in a long time or life-long,causing swine atrophic rhinitis,as well as kennel cough and nasal thiamethoxam of rabbits.Biofilm can enhance the resistance of bacteria to the external environment,and evade the immune defenses mechanisms of the hosts.It is not easy to be removed at the site of infection,leading to persistent infection;and can resist the action of antibiotics,which makes the microbial resistance within the membrane increase one thousand times.And it is about more than 80% of the bacterial diseases process that the bacteria is survival by the state of biofilm.However,there is no clear consenus of the mechanism of biofilm at home and abroad,which need studing father more,at the same time,have not more valuabe protective antigen is found by biofilm.Therefore,this study determined the optimum conditions for biofilm formation through the optimization,and then observed its membrane structure.After that,using comparative proteomics to reveal the mechanism of biofilm formation in protein level,and studying characteristics of the related proteins to provide a scientific basis for the prevention and control of biofilm and vaccine development.1 The biofilm structure observation and optimization in vitro of Bordetella bronchiseptica.In order to determine the conditions of bacterial biofilm forming effects,the study in vitro used qualitative and quantitative analysis to detect differences of one strain of swine Bordetella bronchiseptica producing biofilm in different environments(time,concentration of sodium chloride,pH,glucose,sucrose,lactose)and investigated the structure of biofilm were observed at the same time.The results showed that 24 h was Bb biofilm mature and stable period,the biofilm OD decreased gradually when sodium chloride concentration was more than 0.4%,pH slightly alkaline medium for b.bronchiseptica biofilm growth,lactose can promote the formation of b.bronchiseptica biofilm.It was observed that after 24 h,the grid likebiofilm was formed and stabilized.2 Comparative proteomic analysis between biofilm and planktonic cells of Brodetella bronchisepticaIn this study,we compared the expression difference of the whole bacterial protein between two kinds of state of Brodetella bronchiseptica by a two-dimensional gel electrophoresis(2-DE).The results revealed that there were 15 up-regulated protein spots and 9 down-regulated.Then identified seven up-regulated proteins by mass spectrometry,as a result,the up-regulated protein spots mainly included elongation factor,superoxide dismutase,molecular chaperone,and so on,Mainly acting on the stress,regulation and metabolism.The results deepened the study of bacterial biofilm formation mechanism at the protein level,and provided scientific basis for the prevention and control of biofilm,as well as its in-depth study.3.Cloning,expression and biological characteristics of superoxide dismutase(SOD)geneTo explore the biological characteristics of superoxide dismutase from porcine Bordetella bronchiseptica,this study designed primers referring to SOD gene sequence of RB50 strain in GenBank,and amplified by PCR technique,then cloning into pET-28a(+)vector to construct recombinant plasmid pET-SOD and transforming into E.coli BL21(DE3),inducing by IPTG to express and purify the protein,after that,determinating the enzyme activity of SOD and immunizing mice,At last,using poison attact experiment to detect efficiency,and studied the polyclonal antibody on the inhibition of biofilm.As a conclusion,the expression form of the recombinant protein was more clear,the specific enzyme activity was 1272(u/mgprot),the efficiency of immune protection was 20%,and the Bb biofilm has some inhibitory effect. |
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