基于沙门氏菌PagN外膜蛋白的广谱单克隆抗体的制备与鉴定  

Preparation and Identification of Broad-spectrum Monoclonal Antibody Against Outer Membrane Protein of PagN of Salmonella

作　　者：孟闯[1,2,3] 丁睿清 尚月月 刘钧 徐双媛 康喜龙 焦新安[1,2,3] 潘志明 MENG Chuang;DING Ruiqing;SHANG Yueyue;LIU Jun;XU Shuangyuan;KANG Xilong;JIAO Xinan;PAN Zhiming(Jiangsu Key Laboratory of Zoonoses,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou,Jiangsu 225009;Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality of Ministry of Agriculture and Rural Affairs,Yangzhou University,Yangzhou,Jiangsu 225009)

机构地区：[1]扬州大学,江苏省人兽共患病学重点实验室,江苏扬州225009 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]扬州大学,农业农村部农产品质量安全生物性危害因子(动物源)控制重点实验室,江苏扬州225009

出　　处：《中国家禽》2023年第4期69-75,共7页China Poultry

基　　金：国家自然科学基金项目(32002295);江苏省重点研发计划(现代农业)项目(BE2021354);江苏省政策引导类计划(国际科技合作港澳台科技合作)资助项目(BZ2020013)。

摘　　要：为制备针对沙门氏菌特异性外膜蛋白PagN的单克隆抗体(MAb),研究通过构建重组质粒pColdⅠ-pagN和pGEX-6P-1-pagN,利用大肠杆菌系统表达并纯化了肠炎沙门氏菌His-PagN和GST-PagN重组蛋白,免疫6~8周龄BALB/c小鼠,利用B淋巴细胞杂交瘤技术筛选获得2株能稳定分泌PagN蛋白MAb的杂交瘤细胞株3B3和3F10。结果显示:3B3和3F10的亚型均为IgG2b,ELISA效价分别为1.14×10^(9)和1.32×10^(8),Westernblot分析显示其均能特异性结合His-PagN蛋白;进一步分析显示3B3和3F10可特异性识别9种不同血清型沙门氏菌,而不与9种非沙门氏菌菌株反应;全菌包板间接ELISA结果显示3F10能特异性识别不同血清型沙门氏菌,显示出用于沙门氏菌检测的潜能。研究表明制备的单克隆抗体3B3和3F10可特异性识别不同血清型沙门氏菌PagN蛋白,为沙门氏菌检测提供了重要生物材料。In order to prepare monoclonal antibody(MAb)against Salmonella specific out membrane protein of PagN,the re⁃combinant plasmids pColdⅠ-pagN and pGEX-6P-1-pagN were constructed and then His-PagN and GST-PagN recombinant proteins of Salmonella Enteritidis were expressed and purified by E.coli system.BALB/c female mice aged 6 to 8 weeks were then immunized with the purified His-PagN protein to prepare MAb hybridoma cells by B lymphocyte hybridoma technology.Two cell strains named 3B3 and 3F10 which could stably secret MAb against Salmonella PagN protein were screened.The results showed that both of 3B3 and 3F10 belonged to subtype IgG2b,which of ascite titer was 1.14×10^(9) and 1.32×10^(8),respectively.Western blot results showed that 3B3 and 3F10 could specifically bind to His-PagN protein,indicating their specificity.Both 3B3 and 3F10 could specifically recognize 9 serotypes of Salmonella,but didn′t react with 9 strains of non-Salmonella bacteria.Indirect ELISA coating with whole bacteria showed that 3F10 could specifically recognize different serovars of Salmonella,dis⁃playing the potential for Salmonella detection.The 3B3 and 3F10 prepared in this study could specifically recognize PagN protein of different serotypes of Salmonella,which provided important materials for the detection of Salmonella.

关 键 词：沙门氏菌 PagN 外膜蛋白 单克隆抗体 反应谱 

分 类 号：S855.1[农业科学—临床兽医学]