布鲁氏菌ABC转运蛋白相关基因cydDC缺失株的构建及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2025.11.029

摘要/Abstract

摘要： 【目的】探究ABC转运蛋白相关基因cydDC在布鲁氏菌中的生物学功能，揭示其在应激和细胞内存活方面的作用。【方法】以羊种布鲁氏菌16M为研究对象，利用同源重组技术构建基因缺失株16MΔcydDC，并通过回补质粒转化的方法构建其回补株16M CΔcydDC，通过比较3种菌株的生长特性、应激敏感性、胞内存活能力及炎症因子诱导水平，系统评估cydDC基因的功能。【结果】PCR鉴定结果显示，16M野生株可获得大小为4 504 bp的特异性条带，而缺失株16MΔcydDC和回补株16M CΔcydDC特异性条带大小为1 088 bp，互补株还扩增出大小为3 819 bp的条带，证实菌株构建成功；3种菌株在相同体外培养条件下的生长趋势相似；但体外应激试验结果显示，16MΔcydDC缺失株对酸性(pH 4.0～4.5)和氧化(1 mmol/L H₂O₂)应激的敏感性显著高于野生株和互补株(P＜0.05)。胞内增殖试验和促炎因子mRNA水平表达结果显示，与野生株和互补株相比，在感染24 h时，缺失株16MΔcydDC在RAW264.7细胞内的存活能力显著降低(P＜0.05)，炎症因子白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的表达显著上调(P＜0.05)。【结论】ABC转运蛋白系统相关基因cydDC有利于布鲁氏菌在酸性应激、氧化应激和细胞内环境下存活，通过调控细胞炎症反应，减少此类刺激对细菌的损伤。

关键词: 布鲁氏菌; cydDC基因; 基因缺失; 生物学特性

Abstract: 【Objective】 This study aimed to investigate the biological functions of cydDC gene in Brucella,and reveal its role in stress response and intracellular survival. 【Method】 Using Brucella melitensis 16M as the model organism,cydDC gene deletion strain (16MΔcydDC) was constructed through homologous recombination,and a complemented strain (16M CΔcydDC) was generated by transforming complementation plasmid.By comparison of growth characteristics,stress sensitivity,intracellular survival capacity,and inflammatory cytokine induction among these three strains,the functional role of the cydDC gene was comprehensively evaluated. 【Result】 PCR analysis identified a 4 504 bp amplicon in wild type 16M,while both 16MΔcydDC and 16M CΔcydDC strains showed a 1 088 bp fragment,with the complemented strain additionally exhibiting a 3 819 bp product,confirming successful mutant construction.Although all three strains displayed comparable growth kinetics in vitro,stress susceptibility assays demonstrated significantly enhanced sensitivity of the 16MΔcydDC strain to both acidic (pH 4.0-4.5) and oxidative (1 mmol/L H₂O₂) challenges compared with wild-type and complemented strains (P＜0.05).Intracellular proliferation assays further revealed that at 24 h post-infection, 16MΔcydDC strain showed significantly impaired survival in RAW264.7 macrophages (P＜0.05),concomitant with markedly upregulated expression of proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) (P＜0.05). 【Conclusion】 ABC transporter-related gene cydDC contributed to Brucella survival under acidic and oxidative stress,as well as intracellular environments,and reduced the damage caused by such stimuli to the bacteria by regulating cellular inflammatory responses.

Key words: Brucella; cydDC gene; gene deletion; biological characteristics

中图分类号:

S852.61

姜治强, 张皓博, 刘蒙达, 孙翔翔, 邵卫星, 常璐, 亓菲, 孙世雄, 焉鑫, 南文龙, 孙淑芳, 樊晓旭, 黄保续. 布鲁氏菌ABC转运蛋白相关基因cydDC缺失株的构建及生物学特性分析[J]. 中国畜牧兽医, 2025, 52(11): 5338-5349.

JIANG Zhiqiang, ZHANG Haobo, LIU Mengda, SUN Xiangxiang, SHAO Weixing, CHANG Lu, QI Fei, SUN Shixiong, YAN Xin, NAN Wenlong, SUN Shufang, FAN Xiaoxu, HUANG Baoxu. Construction and Biological Characterization Analysis of Brucella ABC Transporter-related Gene cydDC Deletion Strain[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5338-5349.

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