The Effects On DNA Vaccine Of Murine Zona Pellucida 3 With Interleukin-2

Zona pellucida 3 (ZP3), a major glycoprotein outside the oocyte, possesses the sperm receptor fbnction. The antibodies to ZP3 protein may block sperm/oocyte interaction for population control. However, two major problems currently hinder the development of ZP3 protein vaccine. When some parts of female subjects were immunized with ZP3 vaccine, they developed ovarian histopathology. Becauce mZP3 protein was shown to possess at least two T cell epitopes and the capacity to induce T cell-mediated autoimmune oophoritis. In addition, it was difficult to get enough special ZP3 protein as a vaccine in practice, and the process of such vaccine was very complex. However, It is imperative that potential human or animal contraceptive vaccines be free of serious side effects in populations without genetic restriction, simply produced and conveniently applied in any time.In this experiment, mZP3 gene was cloned into the expression vector pGEX-4T-l for the specific mZP3 protein. The eukaryotic plasmid pcDNA-mZP3+pcDNA3-mIL2 was evaluated through the transient expression of mZP3 and mIL2 gene in eukaryotic expression system. Then I described the design of a cassette, pcDNA-mZP3+pcDNA3 -mIL2, which overcomes the above two problems, maintains the advantage that mZP3 DNA vaccine may elicit the antibody of high level to recognize native mZP3 in BALB/c mice, and reduce the capability of their fertility. After the immunization, the specific antibodies that were induced by mZP3 DNA vaccine in the immunized mice serum were tested by the enzyme-linked immunosorbent assay (ELISA). The mechanism of T cellular response to pcDNA-mZP3 vaccine were explored with the special mRNA level of-5-EL-4 gene and IFN- Y gene in ovary of the immunized mice assayed by SqRT-PCR (Semi-quantitative Reverse Transcription and Polymerase Chain Reaction). In addition to that, ovarian .pathology of the immunized mice and evaluation of the potency of the mice infertility were assessed.The specific stains according to the expection of a by A 76kD mZP3 protein was presented in SDS-PAGE from E.coli BL21. It was indicated that mZP3 protein from E.coli BL21 could be regarded as the antigen to specific antibody in immunized mice.It was evident that the pcDNA-mZP3 be choose as a candidate immunogens, the pcDNA3-mIL2 as an adjuvant, through the effective expression of mZP3 gene in eukaryotic COS-7 cells. The efficacious anti-reproduction reactivity to mZP3 DNA vaccine was brought out in BALB/c mice by the inoculation of sole mZP3 DNA vaccine, but the DNA vaccine caused a severe form of oophoritis. In addition, the cassette, pcDNA3-mZP3 +pcDNA3-mIL2, could efficaciously maintain the high antibody titers in the serum of mice, reduce the capability of their fertility and elicit the development of abnormal follicle in the ovaries as a result of the immune pcDNA3- mZP3. Especially, this cassette could elicit The mRNA expression of IFN- Y gene and IL-4 gene comparatively high in the ovaries and the light injury of ovaries to overcome the concomitant autoimmune oophoritis in the immunized BALB/c mice, the result indicated that the expression of mIL2 gene neutralize T cell-mediated response in immunized mice. The result has still supported the idea that it may still be necessary to research the phenomenon thoroughly for us that the high antibodies induced by mZP3 DNA vaccine have nothing to do with autoimmune oophoritis in the mice or other animals.