| Objective:To analyze the immunocompetence of the LTB-PorB DNA vaccine and recombinant protein LTB-PorB in female BALB/c mice through intranasally immunization and identify weather the mucosal adjuvant LTB could assist PorB induce a more effective mucosal immunologic responses. In addition, in order to improve the vaccine immune efficacy, we want to explore improved immunologic responses induced by a LTB-PorB DNA prime-LTB-PorB protein boost immunization.Methods:1. Recombinant eukaryotic expression recombinant pcDNA3.1(-)/ltB, pcDNA3.1(-)/porB and pcDNA3.1(-)/ltB-porB were identified by Polymerase Chain Reaction(PCR), enzyme digestion and then prepared. Female BALB/c mice were inoculated with purified eukaryotic expression recombinant through intranasally immunization at the weeks 0,2,4,6. 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. 2. Recombinant protein LTB, PorB, LTB-PorB were expressed efficiently in Escherichia coli BL21/(pET-30a/ltB, pET-30a/porB, pET-30a/ltB-porB) in the form of inclusion bodies. The renatured recombinant proteins had antigenicity, which was confirmed by Western blot. Female BALB/c mice were inoculated with renatured recombinant proteins 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.3. A LTB-PorB DNA vaccine (pcDNA3.1(-)/ltB-porB) and recombinant protein LTB-PorB were prepared. Female BALB/c mice were inoculated with a LTB-PorB DNA vaccine followed by boosting with protein LTB-PorB through intranasally immunization at the weeks 0,2,4,6. 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. The levels of PorB specific slgA in genital tract and IgG in serum shown upward trend along with the days post innoculation in pcDNA3.1(-)/ltB-porB group, A450 of sIgA in pcDNA3.1(-)/ltB-porB group was 0.470±0.088 at the week 8, which was significantly higher than controls (P<0.01), and the titer was up to 1:320. A450of serum IgG in pcDNA3.1(-)/ltB-porB group was 0.528±0.106 at the week 8, which was significantly higher than the pcDNA3.1(-)/ltB, pcDNA3.1(-)and the PBS controls (P<0.01), and the titer was up to 1:1280. However, the IgG between pcDNA3.1(-)/ltB-porB group and pcDNA3.1(-)/porB control(A45o:0.511±0.094)had no significant difference (P>0.05). The levels of IL-4, IFN-y induced by splenic lymphocyte and Stimulation index of the splenic lymphocyte in pcDNA3.1(-)/ltB-porB group was significantly higher than the pcDNA3.1(-)/ltB, pcDNA3.1(-)and the PBS controls(P<0.05).2. The levels of PorB specific sIgA in genital tract shown upward trend along with the days post innoculation in LTB-PorB group, A450 of sIgA in LTB-PorB group was 0.663±0.095 at the week 6, which was significantly higher than controls (P<0.01), and the titer was up to 1:1280. A450of serum IgG in LTB-PorB group was 0.598±0.062 at the week 4, which was significantly higher than the LTB and the Solution Buffer controls (P<0.01), and the titer was up to 1:5120. However, the IgG between LTB-PorB group and PorB control (A450:570±0.060) had no significant difference (P>0.05). Stimulation index of the splenic lymphocyte in LTB-PorB group was significantly higher than the LTB and the Solution Buffer controls (P<0.05). But the level of IFN-γinduced by splenic lymphocyte between LTB-PorB group and controls had no significant difference (P>0.05).3. In the LTB-PorB DNA vaccine prime-protein boost group at week 8, level of sIgA in genital tract(A450:1.083±0.179, the titer was up to 1:2560), level of serum IgG(A450:1.023±0.116, the titer was up to 1:20480), levels of IL-4 (357.58±34.44 pg/ml) and IFN-γ(261.85±29.92 pg/ml), both of which were induced by splenic lymphocyte, were significantly higher than controls (P<0.01), and stimulation index(2.12±0.29) of the splenic lymphocyte was also significantly higher than controls (P<0.05). The ratios of serum IgG2a/IgG1 in the DNA vaccine group, recombinant protein group and the DNA vaccine prime-protein boost group were 1.678,0.455 and 0.693 respectively.Conclusion:1.The LTB-PorB DNA vaccine and recombinant protein could induce specific cellullar immunologic response and humoral mmunologic response(especially for mucosal immunologic responses) in female BALB/c mice through intranasally immunization. The mucosal adjuvant LTB could assist PorB to induce a more effective mucosal immune response in the genital tract mucosa of mice.2.The DNA vaccine prime-protein boost immunization regimen could enhance both specific cellullar immunologic responses and humoral immunologic response in BALB/c mice, especially for Th2 humoral immunologic response, compared with a LTB-PorB DNA vaccine or a recombinant protein LTB-PorB immunization alone.
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