Abstract
BackgroundStray dogs are the reservoirs and carriers of rabies and are definitive hosts of echinococcosis. To control the overpopulation of stray dogs, zona pellucida 3 (ZP3), a primary receptor for sperm, is a potential antigen for developing contraceptive vaccines.To enhance the immune responses and contraceptive effects of canine ZP3 (cZP3), dog gonadotropin-releasing hormone (GnRH) and a T cell epitope of chicken ovalbumin (OVA) were selected to construct two fusion proteins with cZP3, ovalbumin-GnRH-ZP3 (OGZ) and ovalbumin-ZP3 (OZ), and their contraceptive effects were evaluated in mice.MethodsThe synthesized DNA sequences of OGZ and OZ were cloned into plasmid pET-28a respectively. The fusion proteins OGZ and OZ were identified by SDS-PAGE and Western blot. Mice were immunized with OGZ, OZ and cZP3, and the infertility rates were monitored. Mice immunized with mouse ZP3 (mZP3) or adjuvant alone were used as positive control and negative control, respectively. cZP3- and GnRH-specific antibodies (Abs) were detected by ELISA. The bindings of the Abs to oocytes were detected by indirect immunofluorescence assay. The paraffin sections of mice ovaries were observed under microscope for analyzing pathological characteristics.ResultsSDS-PAGE and Western blot analyses showed that the two fusion proteins OGZ and OZ were correctly expressed. ELISA results showed that OGZ vaccine induced both cZP3- and GnRH-specific Abs, and OZ vaccine induced cZP3-specific Ab, which lasted for up to 168 days. The levels of follicle stimulating hormone (FSH) and estradiol (E2) in sera were significantly decreased in OGZ immunized mice. Indirect immunofluorescence results showed that Abs induced by cZP3 and mZP3 could bind to the mouse ZP and dog ZP each other. Compared with the adjuvant group, all vaccine immunized groups significantly decreased the fertility rate and mean litter size. Interestingly, the fertility rate in OGZ-immunized group is the lowest, and only 1 mouse out of 10 mice is fertile. Histological analysis of murine ovarian sections indicated that most of the infertile mice in the immunized groups lacked mature follicles as well as accompanied by inflammatory infiltration. Meanwhile, immunization with OGZ decreased the number of corpora lutea in the infertile mice.ConclusionsThe fusion protein OGZ resulted in the lowest fertility rate and the least mean litter size in the immunized mice. OGZ might be a promising antigen for developing a new contraceptive vaccine for stray dog controlling.
Highlights
Stray dogs are the reservoirs and carriers of rabies and are definitive hosts of echinococcosis
Purification and detection of the fusion proteins OGZ and OZ Fusion protein OGZ was combined with canine gonadotropin-releasing hormone (GnRH) (G), canine ZP3 (cZP3) (Z, 23~ 350 aa) and a T-cell epitope of chicken ovalbumin (O)
After these two proteins were expressed in E.coli by IPTG induction, and after proteins purification, the fusion protein OGZ and OZ showed a major band at 46 kDa and 44 kDa on SDS-PAGE gel respectively as expected
Summary
Stray dogs are the reservoirs and carriers of rabies and are definitive hosts of echinococcosis. To control the overpopulation of stray dogs, zona pellucida 3 (ZP3), a primary receptor for sperm, is a potential antigen for developing contraceptive vaccines. Stray dogs in Xinjiang, China, are the reservoirs and carriers of rabies, and they are definitive hosts of echinococcosis. It is urgent to develop effective contraceptive vaccines to control stray dog’s population. Zona pellucida 3 (ZP3), as the primary receptor of sperm on egg cell and an inducer of the acrosome reaction, plays a key role during fertilization [1,2,3,4]. CZP3 of 35~350aa containing the major B-cell epitopes was selected as the basic antigen for immunocontraception
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