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  

Construction of Brucella Effector Protein BspE Gene Deletion Strain and Its Biological Characteristics

作　　者：张思岚 支飞杰 丁剑 宋银娟 郑福英[2,3] 付强 储岳峰[2,3] 史慧君[1] 许健 ZHANG Silan;ZHI Feijie;DING Jian;SONG Yinjuan;ZHENG Fuying;FU Qiang;CHU Yuefeng;SHI Huijun;XU Jian(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;State Key Laboratory for Animal Disease Control and Prevention,College of Veterinary Medicine,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China;Xinjiang Center for Animal Disease Prevention and Control,Urumqi 830000,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]中国农业科学院兰州兽医研究所,兰州大学动物医学与生物安全学院,动物疫病防控全国重点实验室,兰州730000 [3]甘肃省病原生物学基础学科研究中心,兰州730046 [4]新疆维吾尔自治区动物疾病预防控制中心,乌鲁木齐830000

出　　处：《中国畜牧兽医》2024年第12期5128-5137,共10页China Animal Husbandry & Veterinary Medicine

基　　金：甘肃省自然科学基金重点项目(23JRRA560);甘肃省联合科研基金重点项目(23JRRA1517);甘肃省科技重大专项(22ZD6NA001)。

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2060 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。【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 2060 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

关 键 词：布鲁氏菌 效应蛋白BspE 突变株 生物学特性 

分 类 号：S852.61[农业科学—基础兽医学]