Department of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
Microb Pathog. 2023 Dec;185:106417. doi: 10.1016/j.micpath.2023.106417. Epub 2023 Oct 20.
The gram-negative intracellular bacterium Brucella abortus causes bovine brucellosis, a zoonotic disease that costs a lot of money. This work developed a vector vaccine against brucellosis utilizing recombinant L. lactis expressing Brucella outer membrane protein BAB1-0278. Gene sequences were obtained from GenBank. The proteins' immunogenicity was tested with Vaxijen. The target vector was converted into L. lactis after enzymatic digestion and PCR validated the BAB1-0278 gene cloning in the pNZ8148 vector. The target protein was extracted using a Ni-NTA column and confirmed using SDS-PAGE and western blot. After vaccination with the target vaccine, the expression of IgG subclasses was evaluated by the ELISA method. Cytokine production was also measured by the qPCR method in the small intestine and spleen. Lymphocyte proliferation and innate immune response (NLR, CRP, and PLR) were also assessed. Finally, after the challenge test, the spleen tissue was examined by H&E staining. BAB1-0278 was chosen because of its antigenicity score of 0.5614. A 237-bp gene fragment was discovered using enzymatic digestion and PCR. The presence of a 13 kDa protein band was confirmed by SDS-PAGE and western blot. In comparison to the PBS group, mice given the L. lactis-pNZ8148-BAB1-0278-Usp45 vaccine 14 days after priming had substantially greater levels of total IgG, IgG1, and IgG2a (P < 0.001). Also, the production of cytokines (IFN-γ, TNFα, IL-4, and IL-10) indicating cellular immunity increased compared to the control group (P < 0.001). The target group had a lower inflammatory response, morphological impairment, alveolar edema, and lymphocyte infiltration. An efficient probiotic-based oral brucellosis vaccination was created. These studies have proven that the recommended immunization gives the best protection, which supports its promotion.
革兰氏阴性细胞内细菌布鲁氏菌流产引起牛布鲁氏菌病，这是一种人畜共患疾病，耗费大量金钱。本工作利用表达布鲁氏菌外膜蛋白 BAB1-0278 的重组乳球菌开发了一种针对布鲁氏菌病的载体疫苗。基因序列从 GenBank 获得。使用 Vaxijen 测试蛋白质的免疫原性。目标载体在酶消化后转化为乳球菌，并通过 PCR 验证 BAB1-0278 基因在 pNZ8148 载体中的克隆。使用 Ni-NTA 柱提取目标蛋白，并通过 SDS-PAGE 和 western blot 确认。用目标疫苗接种后，通过 ELISA 方法评估 IgG 亚类的表达。还通过 qPCR 方法在小肠和脾脏中测量细胞因子的产生。还评估了淋巴细胞增殖和先天免疫反应（NLR、CRP 和 PLR）。最后，在攻毒试验后，通过 H&E 染色检查脾组织。选择 BAB1-0278 是因为其抗原性评分为 0.5614。通过酶消化和 PCR 发现了一个 237bp 的基因片段。SDS-PAGE 和 western blot 证实存在 13 kDa 蛋白带。与 PBS 组相比，在 priming 后 14 天给予 L. lactis-pNZ8148-BAB1-0278-Usp45 疫苗的小鼠的总 IgG、IgG1 和 IgG2a 水平显著增加（P < 0.001）。此外，与对照组相比，细胞免疫指示细胞因子（IFN-γ、TNFα、IL-4 和 IL-10）的产生增加（P < 0.001）。目标组的炎症反应、形态损伤、肺泡水肿和淋巴细胞浸润较低。创建了一种有效的基于益生菌的口服布鲁氏菌病疫苗接种。这些研究证明，推荐的免疫接种提供了最佳的保护，支持其推广。
