Qashqai Mansoure, Bertan Emrah, Erisen Semiha, Ozbek Tulin, Vural-Korkut Senay
Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Yıldız Technical University, 34220, Esenler, Istanbul, Turkey.
Institute of Science, Yıldız Technical University-Davutpasa Campus, 34220, Esenler, Istanbul, Turkey.
Transgenic Res. 2025 Apr 29;34(1):22. doi: 10.1007/s11248-025-00441-0.
Brucellosis, caused by Brucella species, is a global threat to livestock farming, resulting in economic losses and socio-economic challenges, particularly in rural areas. Despite its impact, no licensed human vaccines are available. Animal vaccination remains the most cost-effective control method, but traditional vaccine production is expensive. Edible vaccines, using plants as bioreactors to produce immunogenic antigens, offer a low-cost alternative by eliminating complex purification processes. This study developed a transgenic plant by expressing the Brucella abortus outer membrane protein OMP25 in tobacco plants. OMP25, a conserved transmembrane protein with high immunogenicity, was cloned into a Gateway pDONR vector via a Boundary Pairing reaction and transferred to a binary destination vector via a Left-Right reaction. The destination vector was introduced into Agrobacterium tumefaciens and subsequently used for Agrobacterium-mediated transformation of tobacco plants. Transgenic plants were selected on media containing kanamycin, and the expression of the transgene was verified through the fluorescence of green fluorescent protein. Microcallus formation and shoot development on selective media confirmed kanamycin resistance and the successful integration of the transgene. After phenotypic selection, genomic DNA was extracted from transgenic plants and analyzed by PCR (Polymerase Chain Reaction) using primers specific to the OMP25 gene. Positive PCR results validated the successful integration of the OMP25 gene into the plant genome. Gene expression was further confirmed at the RNA level through real-time quantitative PCR (qRT-PCR) and at the protein level via Western blot analysis. Future studies will evaluate immune responses in animal models. This approach demonstrates the potential for low-cost, effective vaccines to combat brucellosis, addressing critical economic and public health challenges.
由布鲁氏菌属引起的布鲁氏菌病是对畜牧业的全球威胁，会造成经济损失并带来社会经济挑战，在农村地区尤为如此。尽管其影响重大，但尚无获批的人类疫苗。动物疫苗接种仍然是最具成本效益的控制方法，但传统疫苗生产成本高昂。食用疫苗利用植物作为生物反应器来生产免疫原性抗原，通过省去复杂的纯化过程提供了一种低成本的替代方案。本研究通过在烟草植株中表达流产布鲁氏菌外膜蛋白OMP25培育出了一种转基因植物。OMP25是一种具有高免疫原性的保守跨膜蛋白，通过边界配对反应克隆到Gateway pDONR载体中，并通过左右反应转移到二元目的载体中。将目的载体导入根癌农杆菌，随后用于根癌农杆菌介导的烟草植株转化。在含有卡那霉素的培养基上筛选转基因植株，并通过绿色荧光蛋白的荧光验证转基因的表达。选择性培养基上微愈伤组织的形成和芽的发育证实了卡那霉素抗性以及转基因的成功整合。经过表型筛选后，从转基因植株中提取基因组DNA，并使用针对OMP25基因的引物通过聚合酶链反应（PCR）进行分析。PCR阳性结果验证了OMP25基因成功整合到植物基因组中。通过实时定量PCR（qRT-PCR）在RNA水平以及通过蛋白质印迹分析在蛋白质水平进一步证实了基因表达。未来的研究将评估动物模型中的免疫反应。这种方法证明了低成本、有效疫苗对抗布鲁氏菌病的潜力，解决了关键的经济和公共卫生挑战。
