| The zona pellucida (ZP), which is an extracellular matrix surrounding oocytes, has received considerable attention as a potential target for contraception since its key role in many reproductive process, however, a major problem associated with this infertility is that it is often associated with the ovarian dysfunction characterized by a loss of primordial follicles from the ovary, which has impeded the applications of ZP contraceptive vaccine. Since the site of fertilization in mammalian animals is in the oviduct, if mucosal immunity against ZP3 can be induced at the mucosal surface of oviduct, it can directly block the fertilization and avoid ovarian dysfunction caused by the high anti-ZP antibody levels in serum for systemic immunity.In this study, to investigate the feasibility of ZP DNA vaccine to induce oviduct mucosal immunity to block the fertilization and avoid the disorder and dysfunction of ovaries, the porcine zona pellucida-3 a eukaryotic expression vector was constructed to develop contraceptive DNA vaccines. The cDNA corresponding to porcine zona pellucida-3 a (pZP3 a ), excluding the N-terminal signal sequence and C-terminal transmembrane domain, was cloned in mammalian expression vector pVAX1, and the recombinant plasmid was named as pVAX1-pZP3 . Then in vitro transient expression was established by in vitro transfection of HeLa mammalian cells with pVAXl-pZP3 a plasmid DNA by lipofection. At 48h after transfection, the expression of pZP3 a at mRNA level and protein level were confirmed by RT-PCR and indirect immunofluorescence (IIP) respectively. Furthermore the recombinant pVAX 1 - pZP3 a was encapsulated with chitosan C390 to make nanoparticles. The morphology of chitosan/pVAXl-pZP3 a nanoparticles was determined by Atomic Force Microscope(AFM) and the imaging demonstrated that the diameter range of the particles is from 100nm to 300nm (the mean diameter is 224.6nm) and the shape is spherical or elliptical. The determination of encapsulated efficiency of chitosan/pVAX1- pZP3 a nanoparticles shows that more than 90% pVAXl-pZP3 a DNA are encapsulated with chitosan. Hela cells transfected with Chitosan/pVAX1-pZP3 a nanoparticles can react with anti-pZP3 antibody, showingbright-green fluorescence in the IIF assay, which indicates the expression of recombinant pZP3 a protein in vitro. After 5 days feeding mice with those nanoparticles by gastric larvae, the transcription and expression of pZP3 a were detected successfully in mouse intestine by RT-PCR and indirect IIF respectively. Oral immunization with the Chitosan/pVAX1-pZP3 a nanoparticles can induce significantly a mucosal immune response, characterized with IgA antibody reaction in the mice serum and viganal secretions. The anti-ZP3 IgA antibody titer in the mice serum began to rise at week 2 and increased after boosting at week 4, and the highest titers were attained at week 10-11 then dropped off afterwards. There was also a certain anti-ZP3 IgA titer in the mice viganal secretions. The anti-fertility experiment showed that 5 of 10 immunized mice became non-pregnant when mated with fertility male mice, and the anti-fertility effect was correlative with the anti-ZP antibody titers of the mating. The ovarian sections observation of immunized mice with infertility indicated that the ovarian structure is normal as the control mice, showing the normal follicles development with different stage follicles (primordial follicles, primary follicles, secondary follicles and mature follicles), normal corpus luteum and no lymphocyte filtration.In conclusion, the oral chitosan/pVAX1-pZP3 a plasmid DNA nanoparticles vaccine can induce significantly mucosal immune response and achieve infertility in the absence of any ovarian pathology. Those studies provide new ideas and ways to develop safe, effective, reversible and convenient contraceptive vaccines.
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