布鲁氏菌BPE159基因缺失株的构建及BPE159蛋白对细胞自噬因子表达的影响

doi:10.16431/j.cnki.1671-7236.2023.01.003

摘要/Abstract

摘要： 【目的】构建布鲁氏菌BPE159基因缺失株，研究缺失株体外生长变化特征及其在宿主细胞中的存活能力，探究布鲁氏菌感染期间分泌蛋白BPE159对自噬因子表达的影响。【方法】同源重组方法构建布鲁氏菌BPE159基因重组质粒，电转化布鲁氏菌S2308感受态细胞构建BPE159基因缺失株S2308ΔBPE159。PCR扩增BPE159基因，连接转化构建pBBR1MCS-4-BPE159载体，提取质粒进行电转化，构建BPE159基因回补株S2308ΔBPE159-C。琼脂糖凝胶电泳检测缺失株和回补株遗传稳定性。构建布鲁氏菌感染小鼠巨噬细胞RAW264.7模型，实时荧光定量PCR检测布鲁氏菌侵染后自噬细胞因子ATG5、Beclin1、LC3a和LC3b基因表达水平。以S2308、S2308ΔBPE159和S2308ΔBPE159-C株侵染小鼠巨噬细胞，收集细胞总RNA，实时荧光定量PCR检测BPE159基因缺失对布鲁氏菌侵染后自噬细胞因子表达水平的影响。在相同起始浓度下培养S2308、S2308ΔBPE159及S2308ΔBPE159-C株，观察细菌生长变化趋势；评价S2308ΔBPE159株在不同时间点的生存繁殖能力。【结果】成功构建BPE159基因缺失株S2308ΔBPE159和回补株S2308ΔBPE159-C，并可稳定遗传10代。实时荧光定量PCR结果显示，经布鲁氏菌侵染24 h后，与PBS组相比，S2308组中自噬因子ATG5基因表达水平显著降低（P<0.05），Beclin1、LC3a和LC3b基因表达水平极显著降低（P<0.01）；与S2308组相比，S2308ΔBPE159组自噬因子Beclin1、LC3a基因表达水平显著升高（P<0.05），ATG5、LC3b基因表达水平极显著升高（P<0.01）。生长曲线结果显示，在相同培养条件下，S2308ΔBPE159和S2308ΔBPE159-C株与S2308株具有相似的生长变化趋势。胞内生存试验结果显示，侵染8 h后，S2308ΔBPE159株在细胞中的数量极显著低于亲本株S2308（P<0.01），12和24 h存活能力显著低于S2308株（P<0.05）。【结论】本研究成功构建并获得了具有良好遗传稳定性的布鲁氏菌BPE159基因缺失株和回补株，S2308ΔBPE159株与S2308株生长趋势相似，但生存繁殖能力明显下降；BPE159基因缺失后布鲁氏菌可促进细胞自噬因子的表达，本研究结果为研究布鲁氏菌分泌蛋白的生物学功能和调控机制奠定了基础。

关键词: 布鲁氏菌; BPE159基因; 基因缺失; 自噬

Abstract: 【Objective】 This study was aimed to construct Brucella BPE159 gene deletion strain, and study the in vitro growth characteristics and survival ability of the deletion strains in host cells and explore the effect of the secreted protein BPE159 on autophagy factor expression during Brucella infection.【Method】 The recombinant plasmid of Brucella BPE159 gene was constructed by homologous recombination.The Brucella BPE159 gene deletion strain S2308ΔBPE159 was constructed by electrotransformation of recombinant plasmid into Brucella S2308 competent cells.Specific primers were used to amplify the BPE159 gene, ligated and transformed to construct the pBBR1MCS-4-BPE159 vector, and the plasmid was extracted and electroporated to construct the BPE159 gene complement strain S2308ΔBPE159-C.Genetic stability of deletion and complement strains was determined by agarose gel electrophoresis.The Brucella-infected mouse macrophage RAW264.7 model was constructed, and the expression levels of autophagy cytokines ATG5, Beclin1, LC3a and LC3b genes after Brucella infection were detected by Real-time PCR.Mouse macrophages were infected with S2308, S2308ΔBPE159 and S2308ΔBPE159-C strains, and total RNA was collected.The effect of BPE159 gene deletion on the expression levels of autophagy cytokines after Brucella infection was detected by Real-time PCR.S2308, S2308ΔBPE159 and S2308ΔBPE159-C strains were cultured at the same initial concentration, and the trend of growth changes was observed, and the survival and reproduction ability of S2308ΔBPE159 at different time points was evaluated.【Result】 The BPE159 gene deletion strain S2308ΔBPE159 and the complement strain S2308ΔBPE159-C were successfully constructed, and they were stably inherited for 10 generations.The results of Real-time PCR showed that after 24 h infection of Brucella, compared with PBS, autophagy factor ATG5 gene was significantly decreased (P<0.05), Beclin1, LC3a and LC3b genes were extremely significantly decreased (P<0.01).Compared with S2308 group, the expression levels of autophagic factors Beclin1 and LC3a genes were significantly increased (P<0.05), and the expression levels of ATG5 and LC3b genes were extremely significantly increased (P<0.01) in S2308ΔBPE159 group.The growth curve results showed that, under the same culture conditions, S2308ΔBPE159 and S2308ΔBPE159-C had similar growth trends to S2308.The results of intracellular survival showed that, 8 h after infection, the number of S2308ΔBPE159 in cells was extremely significantly lower than that of parental strain S2308 (P<0.01), and the viability at 12 and 24 h was significantly lower than that of S2308 (P<0.05).【Conclusion】 In this study, we successfully constructed and obtained BPE159 gene deletion strain and complement strain of Brucella with good genetic stability.S2308ΔBPE159 had a similar growth trend as S2308, but its viability and reproduction ability was obviously reduced.After BPE159 gene deletion, Brucella could promote the expression of autophagic factors.The results of this study laid a foundation for studying the biological function and regulatory mechanism of secreted proteins of Brucella.

Key words: Brucella; BPE159 gene; gene deletion; autophagy

中图分类号:

S852.61

魏春燕, 郭嘉, 朱德馨, 张伟, 朱嘉乐, 邓兴梅, 贾思锋, 刘良波, 张辉. 布鲁氏菌BPE159基因缺失株的构建及BPE159蛋白对细胞自噬因子表达的影响[J]. 中国畜牧兽医, 2023, 50(1): 26-36.

WEI Chunyan, GUO Jia, ZHU Dexin, ZHANG Wei, ZHU Jiale, DEN Xingmei, JIA Sifeng, LIU Liangbo, ZHANG Hui. Construction of Deletion Strain of Brucella BPE159 Gene and Effect of BPE159 Protein on Expression of Cellular Autophagy Factors[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 26-36.

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