Preparation Of Anti-SpvB Antibody And The Influence Of SpvB Gene On NF-κB Signaling Pathway

Objective:The virulence factor encoded by Salmonella plasmid virulence gene spv B possesses ADP-ribosyl transferase activity, which is closely related with bacterial virulence. However, its exact effect on host cell function remains elusive. To investigate the molecular mechanisms of spv B gene on bacterial pathogenic, we firstly constructed the prokaryotic plasmid expressing spv B gene and prepare its antibody, which provide a tool for further investigation of the function of spv B. In the second section, Salmonella macrophages infection model and the eukaryotic expression vector of recombinant plasmid p EGFP-N1-Spv B were employed to investigate the effect of spv B gene on NF-kappa B pathway, the results will provide experimental basis for elucidating the virulent mechanism of spv B gene in Salmonella infection.Methods:One. Bioinformatics analysis on spv B gene and the preparation of anti-Spv B antibody1. The function of spv B gene and its encoding protein was predicted by bioinformatics analysis software. The amino acid sequences of high antigenicity and easily expression were selected as a pseudo clone sequence.2. Primers of spv B gene were designed according to spv B gene sequence. The spv B gene which was carried in wild-type Salmonella typhimurium strain was used as template. The objective fragments obtained from PCR amplification were connected into prokaryotic expression vector p ET-28 a. The p ET28a-Spv B plasmid was then transformed into Escherichia coli BL21(DE3), and expression of spv B was induced with IPTG. The expressed proteins were purified by affinity chromatography methodand SDS-PAGE electrophoresis. Healthy mice were immuned with Spv B protein to prepare polyclonal antibody against Spv B. The specificity of the antibody was assayed by western blotting.Two. The influence of spv B gene on NF-κB signaling pathway1. Wild-type S. typhimurium strain STM-WT carrying the spv B genes on virulence plasmid, spv B mutated strain STM-Δspv B and Δspv B-complemented strain STM-c-Δspv B were used as experimental strains. All strains of bacteria were co-cultivated with mouse-derived macrophage-like cell line J774 A.1 to build infection model in vitro. The expression of IκBα and p65 were determined by western blotting;the nuclear translocation of p65 was observed under fluorescence microscope;Caspase-3 activity was assayed by colorimetry; ubiquitination of IκBα was detected by immunoprecipitation.2. He La cells were transiently transfected with p RL-TK plasmid(reference plasmid), p NFκB-luc plasmid(report gene plasmid), p EGFP-N1 plasmid or p EGFP-N1-Spv B plasmid. Transiently transfected cells were interfered with TNF- a.The influence of spv B gene on NF-κB pathway was assayed by dual luciferase reporter gene assay system. He La cells were transiently transfected with p EGFP-N1-Spv B plasmid, and cells were interfered with TNF-α. The expression of IκBα and p65 were determined by western blotting.Results:One. Bioinformatics analysis on spv B gene and the preparation of anti-Spv B antibody1. The prokaryotic expression plasmid p ET28a-Spv B was successfully constructed,and the Spv B protein was purified by affinity chromatography.2. The polyclonal antibody against Spv B was obtained from mice immuned with Spv B protein. The antibody could specificity conjunct with Spv B protein.Two. The influence of spv B gene on NF-κB signaling pathway1. Western blot detection showed that the level of IκBα in STM-Δspv B infected cells was significantly lower than STM-WT infected cells 30 min post infection(P <0.01). There was no difference in the level of Phospho-p65 between STM-Δspv B andSTM-WT infected cells 30 min post infection. The level of Phospho-p65 in STM-Δspv B infected cells was significantly higher than STM-WT infected cells 4 h and 8 h post infection(P < 0.05).2. The nuclear translocation of p65 was viewed under the fluorescence microscope.The level of p65 in nuclear was much higher in STM-Δspv B infected cells 8 h post infection.3. Western blot detection of Caspase-3 activity showed that the Caspase-3 was activated in STM-WT and STM-c-Δspv B infected cells, which was consistent with the result assayed by colorimetry.4. Immunoprecipitation assay showed that with interfere of MG132 24 h before infection, ubiquitination of IκBα was much higher in STM-Δspv B infected cells 4 h post infection. There was no significant difference in the ubiquitination of IκBα among each infected groups without interfere of MG132.5. Dual luciferase reporter gene assay system showed that the activation of NF-κB signaling pathway in cells transiently transfected with p EGFP-N1-Spv B was much lower than cells transfected with p EGFP-N1(P < 0.01). The activation of NF-κB in cells infected with STM-Δspv B was much higher than cells infected with STM-WT and STM-c-Δspv B infected cells 8 h post infection(P < 0.01).6. Western blot detection showed that the level of IκBα in cells transfected with p EGFP-N1-Spv B was higher than cells transfected with p EGFP-N1 and the level of Phospho-p65 in cells transfected with p EGFP-Spv B was lower than p EGFP-N1 transfected cells.(P < 0.01).Conclusions:1. The spv B prokaryotic expression plasmid was successfully constructed. The Spv B protein and anti-Spv B antibody was obtained, which provide a tool for further investigation of the function of spv B.2. spv B gene suppresses the degradation of IκBα and translocation of p65, which inhibit NF-κB signaling pathway.