Immunocontraceptive potential of a GnRH receptor-based fusion recombinant protein

Abstract

Background The management of stray dog population has been of utmost importance due to their overpopulation, increase in dog bites incidence, and rabies. Contraceptive vaccines, a non-surgical alternative to spaying and neutering are viewed as a valuable option for the management of dog population. In this study, the contraceptive potential of a recombinant fusion protein containing the three genes GnRH, GnRH receptor, and ZP3 was explored. Results The gene fragment encoding GnRH, GnRHR, and ZP3 along with the antigenic epitopes of canine distemper virus and tetanus toxoid was assembled, synthesized, and cloned into pET28a expression vector. The resulting construct GVAC08 was successfully transformed into BL21DE3 strain of E. coli and confirmed by colony PCR. The recombinant GVAC08 protein was expressed and purified using Ni-NTA and was confirmed to be a 50-KDa protein by SDS PAGE and Western blot. Mice were immunized with the GVAC08 protein using Freund’s complete adjuvant followed by a booster using Freund’s incomplete adjuvant. This induced a high antibody titer against GnRH, GnRH receptor, and ZP3 which was determined by ELISA. Conclusion Mating studies showed that the GVAC08 recombinant protein was able to reduce the litter size in immunized mice showing improved efficacy. However, the vaccine candidate with further improvements will be a viable contraceptive vaccine.

Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Induces Immune Responses in Mice

“…In line with this, previous studies have shown that antigens containing multiple repeats of ZP3 epitopes stimulate higher antibodies in BALB/c mice compared with those containing single mZP3 epitopes [ 32 , 77 ]. Incorporation of multiple copies of epitopes into antigens has been investigated previously for various weak immunogens to enhance their immunogenicity [ 78 , 79 , 80 ].…”

Plant-produced mouse zona pellucida 3 peptide, its immunogenicity and contraceptive efficacy

“…In line with this, previous studies have shown that antigens containing multiple repeats of ZP3 epitopes stimulate higher antibodies in BALB/c mice compared with those containing single mZP3 epitopes (Sadler et al 2000;Hardy et al 2004). Incorporation of multiple copies of epitopes into antigens has been investigated previously for various weak immunogens to enhance their immunogenicity (Jinshu et al 2004;Haigh et al 2007;Sandam et al 2021).…”

“…containing single mZP3 epitopes(Sadler et al 2000;Hardy et al 2004). Incorporation of multiple copies of epitopes into antigens has been investigated previously for various weak immunogens to enhance their immunogenicity(Jinshu et al 2004;Haigh et al 2007;Sandam et al 2021).Notably, the production of mZP3 antigens in plants seems to increase immunogenicity compared to other production systems. E. coli-produced single mZP3-peptide antigens elicited no signi cant antibody responses to ZP3 in BALB/c immunized mice(Hardy et al 2004), and ZP3 protein expressed in the insect cell-expression system(Hardy 2003) seems to induce lower levels of antibodies against mZP3 peptide compared to our study.…”