b

b. The paraffin sections of mice ovaries were observed under microscope for analyzing pathological characteristics. Results SDS-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 TMUB2 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 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. Conclusions The 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. and were constructed. For recombinant fragment was constructed same as OGZ except for missing the GnRH and the second GGGS (Fig. ?(Fig.1a).1a). The synthesized DNA sequences were codon-optimized for expression, and the expressed fusion proteins were named as OGZ and OZ, respectively. The nucleic acid fragments of and BL21 (DE3) cells respectively. The expressional conditions were optimized by Fluvastatin sodium testing different combination of factors, including isopropyl -D-1-thiogalactopyranoside (IPTG) concentration (0.1, 0.3, 0.5, 0.8, 1, 1.5 and 2?mmol/L), induction time (4?h, 6?h and 8?h) and temperature (25?C, 30?C and 37?C). Finally, the optimal induction conditions in 1?L LB medium for OGZ or OZ expression was as follows: 10?mL overnight culture was inoculated into 1?L LB medium and cultured at 37?C with shaking at 250?r/min. When OD600 reached between 0.4~?0.6, 1?mM IPTG was added into the culture. The culture was continually incubated at 37?C for 4?h. Cell pellets were harvested by centrifugation at 5000?r/min for 15?min and washed once with phosphate buffered saline (PBS). Then, the pellets were resuspended in PBS (100?mg/2?mL, containing 20?L protease inhibitor cocktail) and sonicated in an ice bath for 20?min at 5?s intervals. The cell lysate was harvested by centrifugation at 10000?r/min for 15?min, and the pellets were resuspended in 20?mL binding buffer (20?mM Tris-HCl, pH?7.9, containing 6?M urea, 0.5?M NaCl, and 5?mM imidazole). After spinning at 10000?r/min for 20?min, the filtrated supernatant was passed through a nickel-affinity chromatography column three times and washed 20 Fluvastatin sodium times with a column volume of binding buffer. The fusion proteins were eluted by elution buffer (500?mM imidazole). After ultrafiltration, protein concentrations were determined using a BCA Protein Assay Kit (Thermo). Open in a separate window Fig. 1 Schematic representation and Western blot analysis of the fusion proteins OGZ and OZ. a. The main Fluvastatin sodium components of OGZ were T cell epitope of OVA, GnRH and cZP3. The main components of OZ were T cell epitope of OVA and cZP3. A Fluvastatin sodium flexible linker (GGGS) was put in between the parts. b. OGZ and OZ reacted with antibodies (Abs) against cPZ3. Lane 1: OGZ, lane 2: OZ. C. OGZ reacted with Abs against GnRH. Lane 3: OGZ. Lane M: protein molecular excess weight markers Western blot analysis of the fusion proteins OGZ and OZ The antisera against cZP3 (23~?350 aa) and GnRH.

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