流产布鲁氏菌转录调控因子GntR在小鼠模型上诱导的辅助性T细胞免疫应答

Helper T cell immune responses induced by the Brucella abortus transcriptional regulator GntR in a mouse model

摘要:

[目的] 通过对布鲁氏菌gntR基因进行原核表达，分析其诱导机体产生的Th1和Th2型免疫反应。[方法] 以布鲁氏菌S2308基因组为模板，根据GenBank上公布的S2308 gntR基因序列设计引物，利用分子克隆技术，将gntR基因片段克隆至原核表达载体pET-30a，转化至大肠杆菌BL21感受态细胞中，诱导GntR蛋白表达；利用SDS-PAGE对GntR重组蛋白（rGntR）进行分析；利用AKTAxpress智能多维纯化系统对rGntR进行纯化；利用Western blotting分析其免疫反应性；用rGntR刺激小鼠巨噬细胞RAW 264.7和小鼠脾细胞，利用ELISA试剂盒检测细胞因子IFN-γ、IL-2、IL-4和IL-5，以及IgG抗体的水平。将rGntR免疫小鼠，检测小鼠血清中IFN-γ和IL-4的水平。[结果] gntR基因大小为735 bp，编码245个氨基酸，大约在35 kDa处出现蛋白条带，纯化后为单一条带。WB显示，rGntR具有较好的免疫反应性。rGntR可诱导宿主细胞和小鼠产生较高水平的IFN-γ、IL-4和IgG。[结论] rGntR可在体内或体外诱导辅助性T细胞（Th1和Th2型）免疫反应。本研究可为布鲁氏菌病疫苗的研发提供科学依据。

Abstract:

[Objective] The Th1 and Th2-type immune responses induced by the Brucella gntR gene were analyzed by prokaryotic expression of gntR. [Methods] To clone and express gntR gene of Brucella, a pair of primers for the gntR gene of Brucella S2308 was designed based on published GenBank sequence. The gntR gene was amplified by molecular cloning technology from Brucella S2308 and then cloned into pET-30a vector and transformed to E. coli BL21. The expression of GntR protein was analyzed by SDS-PAGE. The GntR recombinant protein (rGntR) was purified by AKTAxpress intelligent multidimensional purification system. The rGntR immunoreactivity was analyzed by Western blotting. Cytokines IFN-γ, IL-2, IL-4 and IL-5 in murine macrophages (RAW 264.7) and splenocytes stimulated with rGntR were detected by ELISA kit. Mice were immunized with rGntR and IFN-γ, IL-4 and IgG in serum of mice were detected by ELISA kit. [Results] The full-length gntR gene was 735 bp, encoding 245 amino acids. SDS-PAGE showed that rGntR appeared in the approximately 35 kDa position. The band was single after purification. WB indicated that rGntR had good immunoreactivity. The rGntR could induce higher levels of IFN-γ, IL-4 and IgG in host cells and mice. [Conclusion] The rGntR could induce helper T cells (Th1 and Th2-type) immune responses in vivo and in vitro. This study provides scientific basis for the development of brucellosis vaccines.