布鲁氏菌效应蛋白BspE基因缺失株的构建及其生物学特性研究

doi:10.16431/j.cnki.1671-7236.2024.12.002

摘要/Abstract

摘要： 【目的】构建牛种布鲁氏菌(B.abortus)A19株效应蛋白BspE基因缺失株，探究其生长特性及在宿主细胞中的生存、黏附及入侵能力，并观察BspE蛋白的亚细胞定位情况。【方法】以牛种布鲁氏菌A19为研究对象，运用同源重组及SacB反向筛选技术构建布鲁氏菌效应蛋白BspE基因缺失株A19ΔBspE，并通过质粒回补方法构建其回补株A19CΔBspE，比较3种菌株在体外的生长特性；通过平板计数方法分析BspE基因缺失对布鲁氏菌在细胞内的生存及黏附和入侵细胞能力的影响；通过间接免疫荧光试验检测BspE蛋白在RAW264.7细胞中的定位情况。【结果】菌液PCR结果显示，获得大小为2 060 bp的特异性条带，成功构建BspE基因缺失株A19ΔBspE；Western blotting结果显示，获得大小约为14.59 ku的BspE-flag条带，成功构建回补株A19CΔBspE。生长曲线结果显示，在相同的培养条件下，A19ΔBspE与牛种布鲁氏菌A19、A19CΔBspE的生长趋势没有显著差异(P＞0.05)，均在8 h达对数生长期，32 h进入平台期。胞内增殖试验结果显示，在感染48 h内，A19ΔBspE在RAW264.7细胞中的生存能力与牛种布鲁氏菌A19、A19CΔBspE无显著差异(P＞0.05)。细菌黏附和入侵试验结果显示，A19ΔBspE黏附和入侵RAW264.7细胞的能力与牛种布鲁氏菌A19、A19CΔBspE没有显著差异(P＞0.05)。间接免疫荧光试验结果显示，BspE蛋白主要分布于核周区域。【结论】本研究证实了BspE基因的缺失不影响布鲁氏菌在体外的生长及在RAW264.7细胞中的生存、黏附及入侵能力，且 BspE 蛋白主要定位在核周区域，为后续研究布鲁氏菌效应蛋白BspE的生物学功能和致病机制奠定基础。

关键词: 布鲁氏菌; 效应蛋白BspE; 突变株; 生物学特性

Abstract: 【Objective】 The purpose of this study was to construct the BspE gene deletion strain of Brucella abortus (B.abortus) A19 strain,explore the growth characteristics of survival,adhesion and invasion in host cells,and also observe the subcellular localization of BspE protein of B.abortus A19 during infection.【Method】 B.abortus A19 was used as the research object.The Brucella effector protein BspE gene deletion strain A19ΔBspE was constructed by homologous recombination and SacB reverse screening technology,and its complement strain A19CΔBspE was constructed by plasmid complementation method.The growth of three strains was measured and the influence of BspE gene deletion on the survival,adhesion and invasion of Brucella in cells was analyzed.The localization of BspE protein in RAW264.7 cells was observed by indirect immunofluorescence assay.【Result】 PCR results of the bacterial solution showed that a specific band with a size of 2 060 bp was obtained,and the BspE gene deletion strain A19ΔBspE was successfully constructed.Western blotting results showed that a BspE-flag band with a size of approximately 14.59 ku was obtained,and the complementation strain A19CΔBspE was successfully constructed.The growth curve results showed that under the same culture conditions,there was no significant difference among B.abortus A19,A19ΔBspE and A19CΔBspE (P＞0.05),all of them reached the logarithmic growth phase at 8 h and entered the platform phase at 32 h.The results of intracellular proliferation test showed that the viability was not significantly different from B.abortus A19,A19ΔBspE and A19CΔBspE during 48 h post infection in RAW264.7 cells (P＞0.05).The results of bacterial adhesion and invasion test showed that the ability of A19ΔBspE to adhere and invade RAW264.7 cells was not significantly different from that of B.abortus A19 and A19CΔBspE (P＞0.05).The result of indirect immunofluorescence assay showed that BspE protein was mainly distributed in the perinuclear of RAW264.7.【Conclusion】 This study proved that the deletion of BspE gene did not affect the growth of Brucella in vitro and the survival,adhesion and invasion in RAW264.7 cells,and BspE protein was mainly located in the perinuclear region,which laid a foundation for the biological function and pathogenic mechanism of Brucella effector protein BspE.

Key words: Brucella; effector protein BspE; mutant strain; biological characteristics

中图分类号:

S852.61

张思岚, 支飞杰, 丁剑, 宋银娟, 郑福英, 付强, 储岳峰, 史慧君, 许健. 布鲁氏菌效应蛋白BspE基因缺失株的构建及其生物学特性研究[J]. 中国畜牧兽医, 2024, 51(12): 5128-5137.

ZHANG Silan, ZHI Feijie, DING Jian, SONG Yinjuan, ZHENG Fuying, FU Qiang, CHU Yuefeng, SHI Huijun, XU Jian. Construction of Brucella Effector Protein BspE Gene Deletion Strain and Its Biological Characteristics[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5128-5137.

参考文献

[1] 丁雪, 王岩, 刘丽蓉, 等.布鲁氏菌病检测和防治方法研究进展[J].中国动物检疫, 2019, 36(5):43-48.DING X, WANG Y, LIU L R, et al.Research progress on detection and control of brucellosis[J].China Animal Health Insepction, 2019, 36(5):43-48.(in Chinese)
[2] HOU H, LIU X, PENG Q.The advances in brucellosis vaccines[J].Vaccine, 2019, 37(30):3981-3988.
[3] MYENI S, CHILD R, NG W T. et al.Brucella modulates secretory trafficking via multiple type Ⅳ secretion effector proteins[J].PLoS Pathogens, 2017, 9(8):e1003556.
[4] 肖启程, 田一男, 彭广能, 等.布鲁氏菌Ⅳ型分泌蛋白及其参与免疫应答的研究进展[J].中国人兽共患病学报, 2017, 33(11):1029-1035.XIAO Q C, TIAN Y N, PENG G N, et al.Research advance on Brucella Ⅳ secretion system proteins and its immune response[J]. Chinese Journal of Zoonoses, 2017, 33(11):1029-1035.(in Chinese)
[5] 吴同垒, 王真, 单晓枫, 等.羊种布鲁菌效应蛋白的筛选[J].中国兽医学报, 2018, 38(5):924-936.WU T L, WANG Z, SHAN X F, et al.Screening of Brucella melitensis effectors[J].Chinese Journal of Veterinary Science, 2018, 38(5):924-936.(in Chinese)
[6] MA Z, YU S, CHENG K, et al.Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host[J].Journal of Veterinary Science, 2022, 23(1):e8.
[7] JEANBAPTISTE L, JULIE R, SANTOS L T L, et al.The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells[J].Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(32):e2105324118.
[8] 支飞杰.转录调控因子ArsR6调控布鲁氏菌毒力的分子机制研究[D].杨凌：西北农林科技大学, 2022.ZHI F J.Effect of transcriptional regulator ArsR6 on regulation of virulence in Brucella[D].Yangling:Northwest A&F University, 2022.(in Chinese)
[9] DARBARI C V, WAKSMAN G.Structural biology of bacterial type Ⅳ secretion systems[J].Annual Review of Biochemistry, 2015, 84(1):603-629.
[10] GLOWACKA P, ZAKOWSKA D, NAYLOR K, et al.Brucella-virulence factors, pathogenesis and treatment[J].Polish Journal of Microbiology, 2018, 67(2):151-161.
[11] TROKTER M, FELISBERTO-RODRIGUES C, CHRISTIE P J, et al.Recent advances in the structural and molecular biology of type Ⅳ secretion systems[J].Current Opinion Structural Biology, 2014, 27:16-23.
[12] 王玉飞, 陈泽良, 赵红庆, 等.以克隆载体为自杀载体快速构建布鲁氏菌的无痕缺失突变株[J].微生物学通报, 2007, 34(4):642-645.WANG Y F, CHEN Z L, ZHAO H Q, et al.Construction of Brucella unmarked deletion mutant by using conventional cloning vector as suicide plasmid[J].Microbiology China, 2007, 34(4):642-645.(in Chinese)
[13] TIAN X, HUANG L, WANG M, et al.The functional identification of Dps in oxidative stress resistance and virulence of Riemerella anatipestifer CH-1 using a new unmarked gene deletion strategy[J].Veterinary Microbiology, 2020, 247:108730.
[14] 王小凤, 李明奇, 郭嘉, 等.布鲁氏菌vceA基因缺失株的构建及生物学特性研究[J].中国畜牧兽医, 2019, 46(4):986-993.WANG X F, LI M Q, GUO J, et al.Construction and biological characteristics of Brucella vceA gene deletion strain[J].China Animal Husbandry & Veterinary Medicine, 2019, 46(4):986-993.(in Chinese)
[15] 李敏, 史静雪, 李志强, 等.牛种布鲁氏菌2308参考株△VceC基因缺失株的构建与鉴定[J].石河子大学学报(自然科学版), 2017, 35(6):714-719.LI M, SHI J X, LI Z Q, et al.Construction and identification of △VceC gene deletion strain of bovine Brucella 2308 reference strain[J].Journal of Shihezi University (Natural Science), 2017, 35(6):714-719.(in Chinese)
[16] 罗文博, 付梦姣, 焦俊, 等.布鲁氏菌Ⅳ型分泌系统效应蛋白BPE043的功能和致病机制研究[J].中国人兽共患病学报, 2020, 36(8):618-623.LUO W B, FU M J, JIAO J, et al.Functional and pathogenic mechanism of Brucella type Ⅳ secretion system effector BPE043[J].Chinese Journal of Zoonoses, 2020, 36(8):618-623.(in Chinese)
[17] 魏春燕, 郭嘉, 朱德馨, 等.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 C Y, GUO J, ZHU D X, et al.Construction of deletion strain of Brucella BPE159 gene and effect of BPE159 protein on expression of cellular autophagy factors[J].China Animal Husbandry & Veterinary Medicine, 2023, 50(1):26-36.(in Chinese)
[18] 徐朕宇, 邓肖玉, 王月丽, 等.牛种布鲁氏菌A19△BtpA缺失株生物学特性及其免疫原性研究[J].畜牧兽医学报, 2024, 55(5):2135-2145.XU Z Y, DENG X Y, WANG Y L, et al.Biological characteristics of Brucella abortus A19△BtpA deletion strain and its immunogenicity study[J].Acta Veterinaria et Zootechnica Sinica, 2024, 55(5):2135-2145.(in Chinese)
[19] BIALER M G, SYCZ G, MUÑOZ GONZÁLEZ F, et al.Adhesins of Brucella:Their roles in the interaction with the host[J]. Pathogens (Basel, Switzerland), 2020, 9(11):942.
[20] 邓肖玉, 何金科, 杨琴, 等.牛种布鲁氏菌A19 VirB启动子缺失株的构建及其生物学特性研究[J].中国畜牧兽医, 2020, 47(3):864-872.DENG X Y, HE J K, YANG Q, et al.Construction of Brucella abortus A19 virB promoter deletion strain and study on its biological characteristics[J].China Animal Husbandry & Veterinary Medicine, 2020, 47(3):864-872.(in Chinese)
[21] 陈蕾, 张广冻, 李俊玫, 等.布鲁氏菌入侵相关基因IalB缺失株的构建及生物学特性分析[J].微生物学报, 2023, 63(3):1254-1268.CHEN L, ZHANG G D, LI J M, et al.Construction and characterization of IalB-deleted strain of Brucella suis[J].Acta Microbiologica Sinica, 2023, 63(3):1254-1268.(in Chinese)