布鲁菌Omp31的单克隆抗体在流式细胞术中的初步应用  

Preliminary application of monoclonal antibody to Brucella Omp31 in flow cytometry assay

作　　者：杨鑫 田艳君[2] 任慧 胡飞环 张国侠[2] 张辉[4] 王文敬 黎诚耀 Yang Xin;Tian Yanjun;Ren Hui;Hu Feihuan;Zhang Guoxia;Zhang Hui;Wang Wenjing;Li Chengyao(Department of Transfusion Medicine,School of Laboratory Medicine and Biotechnology,Southern Medical University,Guangzhou 510515,China;Department of Infectious Disease,Heilongjiang General Hospital of Agriculture Bureau,Harbin 150088,China;Department of Blood Transfusion,Guangdong Provincial People's Hospital,Guangzhou 510120,China Zhang Guoxia;School of Animal Science,Shihezi University,Shihezi 832003,China)

机构地区：[1]南方医科大学检验与生物技术学院输血医学系,广州510515 [2]黑龙江省农垦总局总医院感染科,哈尔滨150088 [3]广东省人民医院输血科,广州510120 [4]新疆石河子大学动物科学院,832003

出　　处：《中华地方病学杂志》2020年第9期647-653,共7页Chinese Journal of Endemiology

基　　金：国家重点研发计划(2017YFD0500300);校科研启动计划(CX2017N007)。

摘　　要：目的利用布鲁菌Omp31的单克隆抗体,初步建立布鲁菌抗原流式细胞术(FCM)检测方法,分析该方法在人布鲁菌病(简称布病)临床样本检测中的价值。方法牛种布鲁菌2308、104M、S19,羊种布鲁菌M5-90和猪种布鲁菌S2共5个菌株,经超声破碎,获得其菌液上清,采用酶联免疫吸附试验(ELISA)法和蛋白免疫印迹法(Western blot),检测已制备的抗布鲁菌Omp31单克隆抗体5H3对上述5个菌株的识别能力。构建Omp31重组真核表达载体(pcDNA3.1-Omp31)并转染人胚肾细胞(293FT细胞),Western blot检测Omp31蛋白的表达。羊种布鲁菌弱毒疫苗株M5-90侵染人急性单核细胞白血病细胞系(THP-1细胞),构建含菌单核吞噬细胞。将5H3抗体用异硫氰酸荧光素(FITC)或藻红蛋白(PE)标记,采用FCM,利用FITC-5H3抗体检测pcDNA3.1-Omp31转染293FT细胞和含菌单核吞噬细胞,鉴定该抗体对细胞内布鲁菌抗原的识别能力,初步建立FCM的检测方法,并测定FITC-5H3抗体在FCM中的灵敏度。利用双抗体(PE-5H3和FITC-CD14)的流式细胞染色测定人外周血单个核细胞(PBMCs),分析该方法应用于人布病临床检测的可行性。结果抗布鲁菌Omp31单克隆抗体5H3不仅能识别羊种布鲁菌M5-90菌株和猪种布鲁菌S2菌株,还能识别缺失Omp31基因的牛种布鲁菌2308、104M、S19菌株。FITC-5H3抗体可用于流式细胞染色识别细胞内的布鲁菌抗原,表达Omp31的293FT阳性细胞比例约为59.3%,疫苗株M5-90侵染的THP-1阳性细胞比例约为6.2%,检测293FT阳性转染细胞的灵敏度为4%。初步建立了基于PE-5H3和FITC-CD14的双抗体染色的FCM检测方法,该方法检测布病患者PBMCs中的双抗体阳性细胞比例(1.93%)高于健康人群(<0.30%,阴性)。结论利用FCM,初步建立了荧光素标记抗布鲁菌Omp31单克隆抗体5H3检测细胞内布鲁菌抗原的方法,并发现5H3抗体能识别牛种布鲁菌,弥补了布鲁菌抗原检测中Omp31的应用缺陷。此方法的建立为布病病原�Objective Using the monoclonal antibody to Brucella Omp31,flow cytometry(FCM)method for detecting Brucella antigens is established,and to analyze its potential value in clinical diagnosis.Methods The supernatants of sonicated proteins(SSPs)from Brucella abortus(2308,104M and S19),Brucella melitensis(M5-90),and Brucella suis(S2)were identified by Western blotting and enzyme-linked immunosorbent assay(ELISA)with monoclonal antibody(mAb)5H3 to Brucella Omp31,which were prepared by breaking Brucella species with ultra-sonication.The recombinant eukaryotic plasmid(pcDNA3.1-Omp31)was constructed and transfected in 293FT cells,and the expression of Omp31 was detected by Western blotting.THP-1 cells were infected by Brucella melitensis M5-90 strain to simulate mononuclear phagocytes carrying with Brucella spp.To identify the ability of mAb 5H3,FCM for detecting intracellular Brucella was established,mAb 5H3 was labeled with fluorescein isothiocyanate(FITC-5H3)or P-phycoerythrin(PE-5H3),and then the transfected 293FT cells and THP-1 cells invaded by M5-90 strain were individually identified by FCM with FITC-5H3,and sensitivity of FITC-5H3 in FCM was tested.The PBMCs collected from brucellosis patients or normal blood donors were tested by FCM with double mAbs including PE-5H3 and FITC-CD14 to evaluate this method's feasibility in clinical practice.Results MAb 5H3 was able to identify Brucella melitensis(M5-90)and Brucella suis(S2),as well as Brucella abortus(2308,104M and S19)with Omp31 gene deletion.The mAb 5H3 labeled with FITC or PE was used for identifying Brucella antigen in various cells by FCM.The results revealed that the proportion of 293FT positive cells expressing Omp31 was about 59.3%,and the proportion of THP-1 positive cells infected by vaccine strain M5-90 was about 6.2%.In addition,the sensitivity of FCM with FITC-5H3 for the 293FT cells transfected with pcDNA3.1-Omp31 was about 4%.The FCM based on double mAbs staining of PE-5H3 and FITC-CD14 was preliminarily established.For brucellosis patients,the propo

关 键 词：布鲁杆菌属 Omp31 单克隆抗体 流式细胞术 病原学检测 

分 类 号：R516.7[医药卫生—内科学] R446.61[医药卫生—临床医学]