The present study employs the Brucella abortus L7/L12 antigen in a Salmonella secretion platform and investigates its ability to induce protective immune responses against wild type challenge in BALB/c mice. The highly conserved L7/L12 open reading frame was PCR amplified from B. abortus and cloned into a prokaryotic expression vector, pJHL65, directly under the beta-lactamase secretory signal. The plasmid constructs pJHL65::L7/L12 was then transformed into an attenuated Salmonella Typhimurium strain, JOL1800 (∆lon, ∆cpxR, ∆asd, and ∆rfaL), and protein secretion was verified by Western blot. Three mice groups were inoculated with either phosphate-buffered saline (PBS), vector-only control, or the vaccine strain secreting L7/L12 antigen. Assessment of humoral and cell-mediated immune responses revealed successful elicitation of Brucella antigen-specific Th1 and Th2 immune responses that were significantly higher than PBS and vector control groups. The immune responses were confirmed by splenocyte proliferation assay, flow cytometry analysis for CD4+ and CD8+ markers, and RT-PCR based cytokine profiling upon restimulation with L7/L12 purified antigen. Results indicate that immunization with Salmonella secreting L7/L12 antigen demonstrated significant enhancement of cell-mediated immune (CMI) responses in immunized mice. The overall effectiveness of the immunization was evaluated by challenging with virulent B. abortus that revealed significant reduction in Brucella colonization in spleen and liver tissues in Salmonella L7/L12 immunized mice. Delivery of Brucella protective antigen L7/L12 using the Salmonella secretion system can effectively accomplish immunogenic advantages of both Salmonella and L7/L12 to derive robust CMI responses and induce humoral immunity to protect against Brucella infection in the mouse model.

中文翻译：

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由减毒沙门氏菌分泌和递送布鲁氏菌核糖体蛋白L7 / L12组成的活疫苗可诱导体液和细胞免疫反应，并保护小鼠免受流产性布鲁氏菌544攻击。

本研究在沙门氏菌分泌平台中采用了流产布鲁氏菌L7 / L12抗原，并研究了其在BALB / c小鼠中诱导针对野生型攻击的保护性免疫应答的能力。从流产芽孢杆菌PCR扩增高度保守的L7 / L12开放阅读框，并直接在β-内酰胺酶分泌信号下将其克隆到原核表达载体pJHL65中。然后将质粒构建体pJHL65 :: L7 / L12转化为减毒鼠伤寒沙门氏菌菌株JOL1800（Δlon，ΔcpxR，Δasd和ΔrfaL），并通过蛋白质印迹法验证蛋白质的分泌。将三个小鼠组接种磷酸盐缓冲盐水（PBS），仅载体对照或分泌L7 / L12抗原的疫苗株。体液和细胞介导的免疫反应的评估显示成功诱发布鲁氏菌抗原特异性Th1和Th2免疫反应，其明显高于PBS和载体对照组。免疫反应通过脾细胞增殖测定，CD4 +和CD8 +标记的流式细胞术分析以及再用L7 / L12纯化抗原重新刺激后基于RT-PCR的细胞因子分析来确认。结果表明，用沙门氏菌分泌L7 / L12抗原进行的免疫接种可显着增强免疫小鼠的细胞介导的免疫（CMI）反应。通过用有毒力的流产布鲁氏菌攻击，评估了免疫的总体有效性，该菌株显示沙门氏菌L7 / L12免疫小鼠的脾脏和肝组织中布鲁氏菌定植明显减少。