Sandam Nathaniel Philip, Prakash Dhamodhar, Thimmareddy Prashanth
Department of Biotechnology, M.S. Ramaiah Institute of Technology, Bangalore, India.
Geniron Biolabs Pvt. Ltd., Bangalore, India.
J Genet Eng Biotechnol. 2021 May 4;19(1):63. doi: 10.1186/s43141-021-00164-9.
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.
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.
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.
由于流浪狗数量过多、狗咬人事件发生率上升以及狂犬病问题，流浪狗种群的管理至关重要。避孕疫苗作为一种非手术的绝育替代方法，被视为管理狗种群的一种有价值的选择。在本研究中，探索了一种包含GnRH、GnRH受体和ZP3三个基因的重组融合蛋白的避孕潜力。
将编码GnRH、GnRHR和ZP3的基因片段以及犬瘟热病毒和破伤风类毒素的抗原表位进行组装、合成，并克隆到pET28a表达载体中。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进行确认。重组GVAC08蛋白经镍-亚氨基二乙酸（Ni-NTA）表达和纯化，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS PAGE）和蛋白质免疫印迹法（Western blot）确认其为50千道尔顿（KDa）的蛋白。用弗氏完全佐剂对小鼠进行GVAC08蛋白免疫，随后用弗氏不完全佐剂进行加强免疫。通过酶联免疫吸附测定（ELISA）确定，这诱导了针对GnRH, GnRH受体和ZP3的高抗体滴度。
交配研究表明，GVAC08重组蛋白能够减少免疫小鼠的产仔数，显示出更高的效力。然而，经过进一步改进的候选疫苗将成为一种可行的避孕疫苗。
