The Study Of Zona Pellucida DNA Contraceptive Vaccine

Zona pellucida (ZP) is a potential target antigen which is used to develop a progestational contraceptive vaccine. However, the tissue structure disorder and dysfunction of ovaries, which were found in some immunization studies with ZP antigen is a obstacle in developing zona pellucida contraceptive vaccine.To investigate the possibility inducing blocking fertilization anti ZP antibody in oviduct and avoiding interfering the structure and function of ovaries by mucosal immunization with ZP DNA and developing oral ZP DNA contraceptive vaccine, we have constructed a recombinant plasmid pVAXl-pZP3 a , prepared the chitosan immunomicroparticles and studied the expression of the recombinant plasmid in vitro and the capacity to induce mucosal immune response in vivo.A pair of primer was designed depending on porcine ZP3 a (pZP3 a ) cDNA sequence and pVAXl, a expressive vector in mammalian cell. The pZP3 a DNA was amplified by PCR and inserted into pVAXl plasmid. The recombinant plasmid pVAXl-pZP3 a was identified by PCR and double enzyme digesting reaction and DNA sequencing. HeLa cells were transfected with pVAXl-pZP3 a by lipid. The transcript of pZP3 a mRNA in HeLa cells was detected by RT-PCR. Immunofluorescence and Western blot were used to determine the pZP3 a antigen expressed in HeLa cells and its culture medium. Rabbit antiserum against pZP3 was used as the first antibody in Western blot and immunofluorescence. pVAXl-pZP3 a DNA Chitosan immunomicropartices were prepared using chitoan as delivery. Female BALB/c mice were immunized with pVAXl-pZP3 a DNA Chitosan immunomicropartices by oral. Control mice were administered with naked pVAXl-pZP3 a DNA by muscular injection (i.m) or chitosan only by oral. Serum, vaginal flush and oviduct flush were collected for evaluating anti ZP antibody response by ELISA. IgA antibody in oviduct mucosal tissue was determined by immunofluorescence.Results show that the pZP3 a gene was inserted correctly into pVAXl and the recombinant plasmid pVAXl-pZP3 a was constructed successfully. The pZP3 amRNA transcript was found in transfected HeLa cells. Thimbleful pZP3 a antigen was detected by Western blot in culture medium of the HeLa cells transfected with pVAXl-pZP3 a . Bright green fluorescence stimulated by transfected HeLa cells was observed in immunofluorescence analysis. This indicates that HeLa cells transfected with pVAXl-pZP3 a can express pZP3 a protein. Significant specific serum IgA responses were induced in the majority of mice after the oral administration of pVAXl-pZP3 a chitoan immunomicroparticles. And ZP IgA response also was induced in small intestine in some oral imuunized mice, but no specific IgG response was detected in the serum. There was no detectable specific IgA and IgG in control mice. When oviduct tissue sections were analyzed by immunofluorescence, the fluorescence intension of oviduct mucosa in mice that received pVAX1-pZP3 a chitoan immunomicroparticles was significant increased comparing with mice that were administered orally with chitosan, which indicates that the IgA antibody increased in the oviduct of mice immunized with pVAX1-pZP3 a chitoan immunomicroparticles by oral.Results stated above show that zona pellucida DNA contraceptive vaccine was constructed successfully. Specific oviduct mucosal immune responses were induced in mice after oral administration of pVAXl-pZP3 a chitoan immunomicroparticles, which provide some valuable data for further research of zona pellucida DNA vaccine to overcome ovarian dysfunction induced by zona pellucida immunization.