| Objective: To construct a recombinant lactobacillus excreting hCGβ-C3d3, Lb.hCGβ-C3d3, which can be used as a live vaccine via mucosal immunization. To compare anti-hCGβ antibody response induced by reconbinant Lb. expressing hCGβ through different mucosal immunization pathways in different strain mice so as to check the adjuvant effect and mechanisms of C3d3 in mucosal immunization of Lactobacillus live vaccine.Methods: Molecular donning was used to construct plasmids pIlac.hCGβ-C3d3, and then transfected into Lactobacillus casei CECT5276 by electroporation, which could stably express hCGβ-C3d3 protein. Chemoluminescence was used to determine the level of hCGβ in the culture supernatant. The expressed products by the recombinant lactobacillus were analyzed by western blot. The fusion protein hCGβ-C3d3 level was assayed by immunocytochemistry. The 6-8-week-old female BALB/c and C57BL/6 mice were immunized with 108,109,1010 Lb.hCGβ or Lb.hCGβ-C3d3 via oral, nose and vagina, respectively, and boosted with the same dosage 3 weeks later. An indirect ELISA was used to determine anti-hCGβ IgG and IgA antibody titer in vaginal lavage fluid and serum, which were got respectively at the end of 2-8 weeks after the prime immunization. Lymphocytes of were separated. The proliferation of B and T cells in spleen, uterus and vagina were analyzed by flow cytometry. The numbers of antibody-secreting cells of anti-hCGβ IgG or IgA were evaluated by ELI Spot.Results: The plasmid pIlac.hCGβ-C3d3 construction was successful by sequencing. Transfection of the pIlac.hCGβ appeared a higher efficiency than that of pIlac.hCGβ-C3d3, and the highest transfecting efficiency of the both plasmids were got at 1200V and 1000V of the pulse voltage, respectively, i.e. about 1.5×l03CFU/μg DNA and 103CFU/μg DNA. The recombinant Lb. hCGβ and Lb. hCGβ-C3d3 could secrete the protein of interest after 0.5% lactose induction, and the highest level was 31.4 mIU/1010Lb.hCGβ and 39.0 mIU/1010Lb.hCGβ-C3d3. Western blot analysis revealed that the recombinant strains could express the proteins hCGβ or hCGβ-C3d3 with expected molecule sizes. Immunocytochemistry analysis showed that the supernatant obtained from Lb.hCGβ-C3d3 appeared positive staining on Raji cells, while that of Lb.hCGβ was negative staining. The mucosal inoculation by 109,1010 Lb.hCGβ could induce resemble response, while that of 108 only induce lower response. Three differentmucosal immunization pathways could induce likely response. The 109 & 1010 Lb. hCGβ-immunized antiserum after booster could neutralize more than 100ng/ml hCG antigen both in BALB/c and C57BL/6 mice. The highest titers induced by vaginal mucosal immunization were stronger than other pathway in certain conditions. The highest titers induced by Lb. hCGβ-C3d3 were 2-100 times higher than Lb.hCGβ. Antiserum after booster appeared to the biological role of neutralizing hCG antigen. The recombinant Lactobacillus could survival in vagina at least 3 weeks after immunization. Compared to Lb.hCGβ, more proliferation of T and B cells was obtained after mucosal immunization with Lb.hCGβ-C3d3 (p<0.05). The numbers of IgG or IgA antibody-secreting cells in spleen were not significant deviation (p>0.05). While the numbers of IgG or IgA antibody-secreting cells in uterus and vagina were significantly higher induced by Lb.hCGβ-C3d3 than Lb.hCGβ (p<0.05).Conclusions: We have got stable and efficient hCGβ-C3d3 excretion in Lb. casei, which may be induced by 0.5% lactose. Lb.hCGβ and Lb.hCGβ-C3d3 used as live vaccines can induce efficient response through mucosal immunization. Vaginal mucosal immunization may be a better immunization pathway to induce reproductive mucosal anti-hCGβ immunity. C3d3 can enhance humoral immune response in mucosal immunization of Lactobacillus expressing hCGβ, even in different strain mice. Strikingly stimulating the proliferation of T and B cells, increasing the numbers of IgG and IgA antibody-secreting cells might be one of the mechanism of C3d3 enhancing the anti-hCGβ humoral immunity in mucosal immunization.
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