布鲁氏菌分泌蛋白BPE043的功能及其对细胞凋亡基因表达的影响  

Function and Effect on Expressions of Apoptosis-Associated Genes of Brucella Secretion System Effector Protein BPE043

作　　者：王书利 李梦含 高萌萌 王倩 郑好 张荷婷 李志强 李启峰 WANG Shuli;LI Menghan;GAO Mengmeng;WANG Qian;ZHENG Hao;ZHANG Heting;LI Zhiqiang;LI Qifeng(College of Biology and Food,Shangqiu Normal University,Shangqiu 476000,China;Tiankang Biopharmaceutical Co.,Ltd,Urumqi 830011,China)

机构地区：[1]商丘师范学院生物与食品学院,商丘476000 [2]天康生物制药有限公司,乌鲁木齐830011

出　　处：《中国动物传染病学报》2025年第1期21-28,共8页Chinese Journal of Animal Infectious Diseases

基　　金：河南省高等学校重点科研项目(21A230015);河南省重点研发与推广专项(科技攻关)项目(212102310746);河南省绿色功能饲料添加剂开发与应用工程技术研究中心。

摘　　要：本研究旨在分析布鲁氏菌Ⅳ型分泌系统(T4SS)效应蛋白BPE043在布鲁氏菌感染过程和调控细胞凋亡基因表达中的作用。以流产布鲁氏菌2308(S2308)为亲本株,通过同源重组的方法构建布鲁氏菌BPE 043基因缺失株(2308ΔBPE 043)和回补株(2308ΔBPE 043-C)。将亲本株、缺失株和回补株在相同起始浓度下培养,观察它们的生长变化。亲本株、缺失株和回补株感染小鼠,测定小鼠脾脏的荷菌量。亲本株、缺失株和回补株侵染小鼠巨噬细胞RAW 264.7,检测细菌的胞内存活能力。利用实时荧光定量PCR(RT-qPCR)和Western blot技术检测布鲁氏菌侵染后细胞凋亡基因Caspase-3、Caspase-8、Bax和Bcl-2的表达水平。结果显示,亲本株、缺失株和回补株在体外培养的生长趋势基本相同。2308ΔBPE 043感染小鼠脾脏的荷菌量显著低于S2303感染组,且在感染2周后,缺失株感染组脾脏荷菌量的下降趋势较亲本株感染组明显。布鲁氏菌侵染宿主细胞8 h后,2308ΔBPE 043的胞内存活能力显著低于S2308。布鲁氏菌侵染宿主细胞24 h后,2308ΔBPE 043侵染组凋亡基因Caspase-3、Caspase-8和Bax表达水平显著高于S2308侵染组,Bcl-2表达水平显著低于S2308侵染组。2308ΔBPE 043侵染组Caspase-3和Caspase-8的蛋白表达水平,以及Caspase-3和Caspase-8的活性显著高于S2308侵染组。本研究发现,T4SS效应蛋白BPE043在布鲁氏菌感染和胞内存活过程中发挥重要作用,是布鲁氏菌重要的毒力因子,为进一步探究BPE043在布鲁氏菌持续性感染中的作用奠定了基础。This study was aimed to analyze the role of typeⅣsecretion system(T4SS)effector protein BPE043 during Brucella infection and the regulation for expression of apoptosis-associated genes.The Brucella 2308ΔBPE 043 mutant strain and 2308ΔBPE 043-C complementary strain were constructed by homologous recombination based on Brucella abortus 2308(S2308)parental strain.Brucella parental strain S2308,mutant strain 2308ΔBPE 043 and complementary strain 2308ΔBPE 043-C were cultured under the same initial concentration for observation of their growth.Mice were infected with S2308,2308ΔBPE 043 and 2308ΔBPE 043-C respectively and the bacterial loads were tested in their spleen samples.In addition,the murine macrophages RAW 264.7 were infected with S2308,2308ΔBPE 043 and 2308ΔBPE 043-C respectively to determine the bacterial survival capability.The expression levels of apoptosis-associated genes caspase-3,caspase-8,Bax and Bcl-2 were detected by real-time quantitative PCR(RT-qPCR)and Western blot at post infection.The results showed that S2308,2308ΔBPE 043 and 2308ΔBPE 043-C exhibited similar growth rate.In mouse infection model,the bacterial loads in spleens of the mice infected with 2308ΔBPE 043 was significantly lower than that of the mice infected with S2308.The bacterial loads in spleens of the mice infected with 2308ΔBPE 043 reduced sharply after week Ind post infection.Following 2308ΔBPE 043 infection,the mRNA expression levels of caspase-3,caspase-8 and Bax in the RAW 264.7 cells were significantly higher than those in S2308-infected cells while theexpression level of Bcl-2 in the 2308ΔBPE 043-infected cells was significantly lower than that in the S2308-infected cells.The protein expression levels of caspase-3 and caspase-8 and their activities in 2308 BPE043-infected cells were significantly higher than those in S2308-infected cells.These results showed that T4SS effector protein BPE043 playd an important role in Brucella infection and intracellular survival.BPE043 was a virulence-associated factor of B

关 键 词：布鲁氏菌 BPE043 胞内存活 毒力 凋亡 

分 类 号：S852.61[农业科学—基础兽医学]