GUO Xiao,WANG Yuan,FAN Hongjie,et al.Development and epitope mapping of monoclonal antibodies against Salmonella PagC protein[J].Journal of Nanjing Agricultural University,2018,41(5):896-901.[doi:10.7685/jnau.201801033]





Development and epitope mapping of monoclonal antibodies against Salmonella PagC protein
郭晓1 汪远1 范红结12 马喆12
1. 南京农业大学动物医学院/教育部动物健康与食品安全国际联合实验室, 江苏 南京 210095;
2. 重要动物传染病与人畜共患病预防和控制中心江苏协同创新中心, 江苏 扬州 225009
GUO Xiao1 WANG Yuan1 FAN Hongjie12 MA Zhe12
1. College of Veterinary Medicine/Joint International Research Laboratory of Animal Health and Food Safety, Ministry of Education, Nanjing Agricultural University, Nanjing 210095, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
SalmonellaPagCmonoclonal antibodysynthetic peptidespecificity
[Objectives]In order to establish the blocking ELISA(bELISA) Salmonella antibody detection kit,we select and identify a monoclonal antibody(mAb) production hybridoma cell line,which can secret mAb against conserved motif of protein PagC in Salmonella. Moreover,we have detected the specificity and binding epitope of this mAb.[Methods]Immunize BALB/c mouse with purified recombination PagC protein,and produce hybridoma cell line. Identify the conserve domain of PagC and synthesize peptide P1 and P2 according to these domains. Screen mAb with the conserve domain peptides and identify their binding epitope. Western blot was used for the mAb specificity identification.[Results]The relative molecular weight of the recombination PagC protein is 23×103. Six mAb secret hybridoma cell lines were identified(hybridoma cell line). The mAbs of these 6 cell lines were all able to bind to PagC. We coupled peptides P1 and P2 with KLH separately and found that mAb from cell line J could bind to P1. The mAb J was very specific to PagC of Salmonella. It cannot react with PagC from Proteus species,Escherichia coli O1,Shigella sonnei.[Conclusions]The mAb identified in this research has great reaction activity and specificity,and the recognized epitope is located in conserve domain of PagC protein. This mAb is an excellent candidate for construction of blocking ELISA(bELISA) Salmonella antibody detection kit.


[1] 林忠兰,吴兴美,吴光渠. 一起肠炎沙门菌食物中毒事件调查[J]. 海峡预防医学杂志,2017,23(2):89-91. Lin Z L,Wu X M,Wu G Q. Investigation of a case of Salmonella enteritis with food poisoning[J]. Strait Journal of Preventive Medicine,2017,23(2):89-91(in Chinese).
[2] Dailey N,Niemeier D,Elkhoraibi C,et al. Descriptive survey and Salmonella surveillance of pastured poultry layer farms in California[J]. Poultry Science,2017,96(4):957-965.
[3] Wales A D,Davies R H. A critical review of Salmonella typhimurium infection in laying hens[J]. Avian Pathol,2011,40(5):429-436.
[4] Charles S D,Sreevatsan S,Bey R F,et al. A dot immunobinding assay(dot-ELISA)for the rapid serodiagnosis of Salmonella enteritidis infection in chickens[J]. Journal of Veterinary Diagnostic Investigation,1996,8(3):310-314.
[5] Minga U M,Wray C,Gwakisa P S. Serum,disk and egg ELISA for the serodiagnosis of Salmonella gallinarum and Salmonella enteritidis infections in chickens[J]. Scandinavian Journal of Immunology,1992,36:157-159.
[6] Dalby T,Strid M A,Beyer N H,et al. Rapid decay of Salmonella flagella antibodies during human gastroenteritis:a follow up study[J]. Journal of Microbiol Methods,2005,62(2):233-43.
[7] Strid M A,Dalby T,Krogfelt K A,et al. Kinetics of the human antibody response against Salmonella enterica serovars enteritidis and typhimurium determined by lipopolysaccharide enzyme-linked immunosorbent assay[J]. Clinical and Vaccine Immunology,2007,14(6):192-197.
[8] Ma Z,Yang X Y,Fang Y Z,et al. Detection of Salmonella infection in chickens by an indirect enzyme-linked immunosorbent assay based on presence of PagC antibodies in sera[J]. Foodborne Pathogens and Disease,2018,15(2):109-113.
[9] Heffernan E J,Harwood J,Fierer J,et al. The Salmonella typhimurium virulence plasmid complement resistance gene rck is homologous to a family of virulence-related outer membrane protein genes,including pagC and ail[J]. Journal of Bacteriology,1992,174(1):84-91.
[10] Chen J S,Ye H M,Yi Y,et al. Preparation of chloramphenicol artificial immunogen and monoclonal antibodies[J]. Biotechnology & Biotechnological Equipment,2011,25(1):2284-2289.
[11] 付明娟,林接玉,谢捷明. 包涵体蛋白复性的研究进展[J]. 医学综述,2015,21(20):3657-3659. Fu M J,Lin J Y,Xie J M. Progress in the study of inclusion body protein refolding[J]. Medical Recapitulate,2015,21(20):3657-3659(in Chinese).
[12] 包义风,应莲芳,蒋琳. 包涵体蛋白复性技术研究进展[J]. 微生物学免疫学进展,2012,40(2):84-88. Bao Y F,Ying L F,Jiang L. Research progress in renaturation of inclusion body protein[J]. Progress in Microbiology and Immunology,2012,40(2):84-88(in Chinese with English abstract).
[13] Kohler G,Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Journal of Immunology,2005,174(5):2453-2455.
[14] 钱珊珊. 沙门菌SpiC蛋白单克隆抗体的研制与初步应用[D]. 扬州:扬州大学,2015. Qian S S. Development and primary application of monoclonal antibodies against Salmonella SpiC protien[D]. Yangzhou:Yangzhou University,2015(in Chinese with English abstract).
[15] 康孟佼,朱春红,蒋颖,等. 肠炎沙门菌间接ELISA检测方法的建立[J]. 中国家禽,2010,32(15):22-25. Kang M J,Zhu C H H,Jiang Y,et al. Dvelopment of an indirect enzyme-linked immunosorbent assay for detection of Salmonella enteritidis[J]. China Poultry,2010,32(15):22-25(in Chinese with English abstract).
[16] 孙海凤. 猪伪狂犬病病毒gB蛋白B细胞表位解析及其阻断ELISA抗体检测方法的建立[D]. 南京:南京农业大学,2015. Sun H F. Evaluation of B-cell epitopes of gB protein and development of blocking ELISA with monoclonal antibody for detection of antibody to PRV[D]. Nanjing:Nanjing Agricultural University,2015(in Chinese with English abstract).
[17] 杨香林. 猪圆环病毒2型单克隆抗体阻断ELISA方法的建立与核酸疫苗重组体的构建及免疫特性研究[D]. 南京:南京农业大学,2012. Yang X L. Development of blocking ELISA with monoclonal antibody for detection of antibody to PCV2 and immunogenicity of DNA vaccine based on the modififd ORF2 gene[D]. Nanjing:Nanjing Agricultural University,2012(in Chinese with English abstract).
[18] Cho H J,Entz S C,Deregt D,et al. Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA[J]. Canadian Journal of Veterinary Research,2000,64(1):38-43.
[19] Fu Y F,Li P H,Cao Y M,et al. Development of a blocking ELISA using a monoclonal antibody to a dominant epitope in non-structural protein 3A of foot-and-mouth disease virus,as a matching test for a negative-marker vaccine[J]. PLoS One,2017,12(1):550-559.


 WANG Lei,ZHAO Xia,WANG Wenjing,et al.Detection of CRISPR and cas expression analysis in induced drug resistance of Salmonella[J].Journal of Nanjing Agricultural University,2018,41(5):370.[doi:10.7685/jnau.201701021]


更新日期/Last Update: 1900-01-01