沙门菌PhoN蛋白单克隆抗体及其免疫磁珠的制备  

Preparation of monoclonal antibodies and its immunomagnetic beads against Salmonella PhoN protein

作　　者：孟闯[1,2,3] 高杨 刘钧 徐双媛 丁睿清 康喜龙 潘志明[1,2,3] 焦新安 MENG Chuang;GAO Yang;LIU Jun;XU Shuang-yuan;DING Rui-qing;KANG Xi-long;PAN Zhi-ming;JIAO Xin-an(Jiangsu Key Laboratory of Zoonoses,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China;Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality,Ministry of Agriculture and Rural Affairs,Yangzhou University,Yangzhou 225009,China)

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]扬州大学江苏省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：《中国预防兽医学报》2023年第3期287-294,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2022YFC2604203);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项目(BZ2020013)。

摘　　要：为制备针对沙门菌Pho N蛋白的单克隆抗体(MAb),并以其制备沙门菌的免疫磁珠,本研究构建了重组原核表达质粒p Cold I-pho N和p GEX-6P-1-pho N,利用大肠杆菌系统表达并通过亲和层析纯化了鼠伤寒沙门菌重组蛋白His-Pho N和GST-Pho N,分别作为免疫原和筛选抗原,利用B淋巴细胞杂交瘤技术,经间接ELISA方法筛选获得5株能稳定分泌Pho N蛋白MAb的杂交瘤细胞株,分别为8H2、11E5、4B2、8F2、3B2。采用亚类鉴定试剂盒、间接ELISA和western blot方法分别鉴定各MAb的亚型、效价及特异性结合沙门菌Pho N蛋白的能力,结果显示,5株MAb的亚型均为Ig G2a,测定其效价达1∶4096000以上,均能特异性结合His-PhoN和GST-Pho N,而不与His-OmpC、His-OmpF蛋白反应,表明MAb特异性较好。选取4B2 MAb进一步分析其对不同血清型沙门菌和非沙门菌的特异性,western blot结果显示4B2 MAb仅特异性识别不同血清型沙门菌,而不与大肠杆菌、志贺杆菌、空肠弯曲菌和单核细胞增生李斯特菌反应;以全菌作为包被抗原的间接ELISA检测结果也显示4B2 MAb可特异性结合沙门菌。将4B2 MAb偶联Fe3O4羧基磁性纳米微球制备免疫磁珠,通过菌落计数分析其对不同血清型沙门菌和非沙门菌的捕获效率,结果显示该免疫磁珠对鼠伤寒等5种不同血清型沙门菌的捕获率达70%以上,对非沙门菌捕获率低于7%。本研究制备了可特异性识别不同血清型沙门菌的Pho N蛋白MAb及其免疫富集磁珠,为样品的快速预处理和沙门菌检测提供了重要生物材料。To prepare monoclonal antibodies(MAb)against Salmonella PhoN protein and to generate immunomagnetic beads based on the antibodies,the recombinant plasmids pCold I-phoN and pGEX-6P-1-phoN were constructed and then the His-PhoN and GST-PhoN recombinant proteins of Salmonella typhimurium were expressed and purified by affinity chromatography using E.coli system.The His-PhoN and GST-PhoN protein was used as immunogen and screening antigen respectively to prepare hybridoma cells using lymphocyte hybridoma technology.Five cell strains named 8H2,11E5,4B2,8F2,and 3B2,which could stably secrete MAb against PhoN protein were screened by indirect ELISA assay.The isoform,titer and specific binding ability of each MAb to Salmonella were identified by indirect ELISA and western blot.Indirect ELISA analysis showed that the isoforms of the five MAb strains were all IgG2a,and their titers were more than 1∶496000.All MAbs could specifically bind His-PhoN and GST-PhoN,but did not react with His-OmpC and His-OmpF proteins,indicating that MAb had good specificity.The 4B2 MAb was selected for further analysis of its specificity against different serotypes of Salmonella and strains of non-Salmonella.Western blot results showed that 4B2 MAb only specifically recognized different serotypes of Salmonella,and did not react with Escherichia coli,Shigella,Campylobacter jejuni and Listeria monocytogenes.The results of indirect ELISA coating with whole bacteria also showed that 4B2 MAb could specifically bind cells of Salmonella.The immunomagnetic beads were prepared by coupling the MAb with the nanospheres Fe3O4 and the specific trapping ability of the immunomagnetic beads against Salmonella was analyzed.The capture efficiency of the immunomagnetic beads of 4B2 for different serotypes of Salmonella and strains of non-Salmonella was analyzed by colony counting.The result showed that capture rate of the immunomagnetic beads against Salmonella Typhimurium and other five different serotypes was more than 70%,and the capture rate of non-Salmonel

关 键 词：沙门菌 Pho N蛋白 单克隆抗体 特异性 免疫磁珠 

分 类 号：S852.61[农业科学—基础兽医学]