| The hCGβï¼based contraceptive vaccine has been verified to be safe, effective and reversible, while the total efficiency was only 80%-85%. More effective adjuvant or appropriate method should be developed to induce enough antibodies at the mucosal site. It has been proved that immunization through mucosa is able to induce higher titre of antibodies at the reproductive mucosa site. In addition, lactobacillus, as a normal flora of human mucosa, could be used as a vector to express the antigen of interest. In order to testify the possibility of vaccination with lactobacillus expressing antigen of interest through mucosal immunization, we constructed a lactobacillus continually expressing hCGβ antigen. Then we inoculated this strain to the BALB/c murine vagina to investigate the hCGβ antigen production from the recombinant lactobacillus as well as the anti- hCGβ antibody at the vaginal mucosa. The 3H-TdR and ELISA were used respectively to determine the proliferating response to hCG antigen and cytokines produced by the lymphocytes in the spleens of the immunized mice. Section I: Excreting in vitro of hCGβ in LactobacillusObjective: To construct the plasmid pIlac-hCGβ to secret continually the hCGβ protein in Lactobacillus casei (Lb. casei).Methods: The cDNA of hCGβ was ligated to the signal peptide sequence of S-layer protein from Lactobacillus brevis (Lb. brevis) and then cloned into down-stream of the lactose-inducible promoter of an integrative plasmid pIlac. After electroporation into Lb. casei CECT5276, radio immuno-assay (RIA) was used to determine the level of hCGβ in the culture supernatant and cell lysates, and PCR using the genomic DNA of the recombinant lactobacillus as template was performed to confirm whether the hCGβ genehad been integrated successfully into the lactobacillus genome. The hCGβ protein secreted by lactobacillus was then confirmed by Western blotting. After having passaged for 50 generations in MRS without Erythromycin, the hCGβ in the culture supernatant was also analyzed by RIA. Then the pH of the culture medium and the concentration of lactose and glucose in the culture medium were adjusted to achieve the most efficient expression of the protein of interest. Results: Sequencing of the PCR product amplified from the genomic DNA of the recombinant strain indicated that the hCGβ cDNA had been integrated successfully into the genome of Lb. casei CECT5276. The highest concentration of hCGβ in the culture medium amounted to 440mIU/ml in 21 hours after lactose induction. About 78.5% of the objective protein was excreted into the supernatant. The recombinant strain was passaged in MRS with no Erythromycin for 50 generations, and could secrete continually the protein of interest. In western blotting analysis of the recombinant strain culture supernatant, there was a 22kDa protein band, which was concord with the hCGβ protein. When the pH of culture medium was controlled at around 7.0, and the concentration of lactose was 0.75%, hCGβ could be secreted at the highest efficiency. Conclusions: The hCGβ protein has been secreted continually in Lactobacillus casei. Key words:hCGβ, Lactobacillus casei, Secreting expressionSection II: Lactobacillus expressing hCGβ induced antigen-specific antibodies in vagina of BALB/c mice by vaginal immunization.Objective: To produce anti-hCGβ antibody in vagina by inoculating to BALB/c murine vagina with the recombinant lactobacillus expressing the hCGβ protein. Methods: After identifying the hCGβ secreted by COS-7 cell with Western blotting assay, intramuscular immunization of pCMV4-hCGβ was performed to 8 weeks-old BAL B/c mice. ELISA was used to detect the anti-hCGβ antibody in the serum. We then inoculated the lactobacillus expressing hCGβ to the vagina of BALB/c mice. RIA was used to determine the hCGβ level produced by the lactobacillus in the murine vagina. ELISA was used to determine the anti- hCGβ IgA antibodies in the vaginal lavage of mice after the mice were...
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