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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.
CONCLUSIONS: 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.
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.
CONCLUSIONS: 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.
摘要：
背景：由于流浪狗的数量过多，对流浪狗的管理至关重要，增加狗咬伤的发生率，狂犬病。避孕疫苗,一种非手术的替代方法被认为是管理狗种群的一种有价值的选择。在这项研究中,含有三个基因GnRH的重组融合蛋白的避孕潜力，GnRH受体,并对ZP3进行了探索。
结果：编码GnRH的基因片段，GnRHR,ZP3与犬瘟热病毒和破伤风类毒素的抗原表位一起组装，合成，并克隆到pET28a表达载体中。将所得构建体GVAC08成功转化到大肠杆菌BL21DE3菌株中,并通过菌落PCR确认。使用Ni-NTA表达和纯化重组GVAC08蛋白,并通过SDSPAGE和蛋白质印迹确认为50-KDa蛋白。使用弗氏完全佐剂用GVAC08蛋白免疫小鼠,随后使用弗氏不完全佐剂进行加强。这诱导了针对GnRH的高抗体滴度，GnRH受体,和通过ELISA测定的ZP3。
结论：交配研究表明，GVAC08重组蛋白能够减少免疫小鼠的产仔数，显示出改善的功效。然而,进一步改进的候选疫苗将是一种可行的避孕疫苗。
结果：编码GnRH的基因片段，GnRHR,ZP3与犬瘟热病毒和破伤风类毒素的抗原表位一起组装，合成，并克隆到pET28a表达载体中。将所得构建体GVAC08成功转化到大肠杆菌BL21DE3菌株中,并通过菌落PCR确认。使用Ni-NTA表达和纯化重组GVAC08蛋白,并通过SDSPAGE和蛋白质印迹确认为50-KDa蛋白。使用弗氏完全佐剂用GVAC08蛋白免疫小鼠,随后使用弗氏不完全佐剂进行加强。这诱导了针对GnRH的高抗体滴度，GnRH受体,和通过ELISA测定的ZP3。
结论：交配研究表明，GVAC08重组蛋白能够减少免疫小鼠的产仔数，显示出改善的功效。然而,进一步改进的候选疫苗将是一种可行的避孕疫苗。
