Immunocompetence Of A LTB-PorB Recombinant Protein Vaccine Adsorbed By Nano-aluminum Hydroxide

Objective:To use nano-aluminum hydroxide adsorbing recombinant protein PorB and LTB-PorB and to analyze the immunocompetence of them in female BALB/c mice through intranasally immunization and identify whether the nano-aluminum hydroxide could promote recombinant protein PorB and LTB-PorB induce more effective humoral immunity response and cell immunity response, especially more efficient mucosal immunologic response. we want to carry out beneficial exploration and provide the evidence of experimentation through mucosa path infected pathogen.Methods:1. The particle of nano-aluminum hydroxide was prepared by microemulsion. the particle size was measured by Nano-Z90.It confirmed that it was asepsis for the organism through toxicity of animal and cell;2. Recombinant protein LTB,PorB,LTB-PorB were expressed efficiently in Escherichia coli BL21/ (pET-30a/LTB-PorB, pET-30a/porB, pET-30a/LTB) in the form of inclusion bodies. The renatured recombinant proteins had antigenicity, which was confirmed by SDS-PAGE and Western blot. enzyme digestion and then prepared;3. To use the adsorption trial to analysis the stability of nano-aluminum hydroxide and aluminum hydroxide adsorbing recombinant protein;4. Female BALB/c mice were inoculated with the vaccine that nano-aluminum hydroxide adsorbing Recombinant protein PorB and LTB-PorB without endotoxin through intranasally immunization at the weeks 0, 2, 4. Next, humoral immunologic response and cellullar immunologic response were detected in female BALB/c mice by enzyme linked immunosorbent assay(ELISA) and methyl thiazolyl tetrazolium(MTT) colorimetric assay. Results:1. One diameter of 100-200nm nano-aluminum hydroxide was procured .They was measured by Nano-Z90. By analysis with toxicity of animal and cell, nano-aluminum hydroxide was availible;2. The levels of PorB specific sIgA in genital tract and IgG in serum shown upward trend along with the days post innoculation in Nano-P and Nano-L-P groups,A450 of IgG in Nano-P and Nano-L-P groups respective were 0.724±0.076,0.854±0.146 at the week 6,which were statistical significance than controls (P<0.01)(PBSgroup,Nano group,LTB group,PorB group,LTB-PorB group);A450 of sIgA in Nano-p and Nano-L-P groups respective were 0.487±0.052,0.637±0.052at the week 6, which were statistical significance than the others (P<0.01);The levels of IL-4 induced by splenic lymphocyte in Nano-P and Nano-L-P groups were statistical significance than the others(P<0.05);the level of IFN-γinduced by splenic lymphocyte between Nano-P group, Nano-L-P group and the other groups had no statistical significance (P>0.05);There was no statistical significance between Nano-P and Nano-L-P groups (P>0.05);The ratio of IgG1 and IgG2a was shown that nano-aluminum hydroxide adsorbing recombinant was main induced humoral immunity responses.Conclusion:1.The vaccine that nano-aluminum hydroxide adsorbing recombinant protein could induce specific humoral mmunologic response(especially for mucosal immunologic responses) in female BALB/c mice through intranasally immunization. nano-aluminum hydroxide could assist PorB and LTB-PorB to induce a more effective mucosal immune response in the genital tract mucosa of mice.