【作者】 王奔奔； 牟路萌； 王茜； 赵姗姗； 张欢； 王震； 陈创夫； 王远志；

【Author】 Wang Benben;Mou Lumeng;Wang Qian;Zhao Shanshan;Zhang Huan;Wang Zhen;Chen Chuangfu;Wang Yuanzhi;College of life sciences,Shihezi University;The Central Hospital of Shandong Energy Zibo Mining Group;School of Medicine,Shihezi University;School of Animal Science and Technology,Shihezi University;

【机构】 石河子大学生命科学学院； 山东能源淄博矿业集团中心医院； 石河子大学医学院； 石河子大学动物科技学院；

【摘要】 目的制备布鲁菌VirB5蛋白单克隆抗体,用于布鲁菌病病原诊断研究。方法用纯化的布鲁菌VirB5蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,经三次筛选及克隆化,建立可以稳定分泌抗VirB5单克隆抗体的杂交瘤细胞株;将杂交瘤细胞对Balb/c小鼠进行腹腔注射,收集腹水,用酶联免疫吸附试验(ELISA)、蛋白免疫印迹(Western blot)分别对获得的单克隆抗体特性进行鉴定。结果获得6株单克隆抗体细胞株(2#、12#、19#、25#、31#和40#),Western Blot显示单克隆抗体细胞株19#能特异性识别VirB5纯化蛋白,并鉴定为Ig G1亚型;间接ELISA法测得腹水抗体效价为1:51200。结论成功获得布鲁菌表面蛋白VirB5的单克隆抗体,该单克隆抗体的制备为布鲁菌病病原诊断方法的建立提供了一种备选材料。更多还原

【Abstract】 Objective To prepare and identify the monoclonal antibody(m Ab) against Protein VirB5 of B. melitensis and which is used to diagnostic research for Brucellosis. Methods Isolating the spleen cells from BALB/C mice after immunized with protein VirB5, and then fuging with the SP2/0 cells. Obtaining the hybridoma cell strain screeting anti-VirB5 m Ab after three rounds of screening and cloning. The hybridoma cells were injected intraperitoneally into Balb/c mice and ascitic fluid was collected. The specificity, titer, immunoglobulin subtype and affinity of the m Ab were evaluated by Enzyme Linked Immunosorbent Assay(ELISA)and Western Blot. Results Six cell strains of hybridoma were obtained and named as 2#, 12#, 19#, 25#, 31# and 40#. Western Blot shown that 19# monoclonal antibodies identified as Ig G1 subtype can specifically recognize protein VirB5. The m Ab titer in ascitic fluid was 1:51200 as determined with ELISA. Conclusion The success in anti-VirB5 m Ab preparation provides an alternative material to establishment of diagnostic methods for brucellosis.更多还原