产单核细胞李氏杆菌内化素G单克隆抗体的制备及双抗体夹心ELISA方法的建立  

Preparation of Monoclonal Antibodies against Internalin G of Listeria monocytogenes and Their Preliminary Application

作　　者：马金锐 史文静 田常青 董志杰 赵学慧 芝吉 曹青 魏衍全 宋维丽 薛惠文[1] 苟惠天[1] MA Jinrui;SHI Wenjing;TIAN Changqing;DONG Zhijie;ZHAO Xuehui;ZHI Ji;CAO Qing;WEI Yanquan;SONG Weili;XUE Huiwen;GOU Huitian(College of Veterinary Medicine Gansu Agricultural University,Lanzhou 730070,China;Animal Husbandry and Veterinary Station,Longmen Town,Lintao County,Lintao 730500,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]临洮县龙门镇畜牧兽医站,临洮730500

出　　处：《畜牧兽医学报》2024年第9期4069-4076,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(31960726,32060822,31560700);甘肃省自然科学基金(21JR7RA482);甘肃农业大学青年导师扶持基金项目(GAU-QDFC-2020-10);甘肃省重点研发计划项目(20YF8FA136);2022年现代丝路寒旱农业科技支持项目(GSLK-2022-17);张家川揭榜挂帅项目(ZC-STK-2023A-030)。

摘　　要：本研究旨在建立一种快速检测产单核细胞李氏杆菌的方法。通过PCR扩增产单核细胞李氏杆菌(LM)内化素G(InlG)基因,构建pET-32a-InlG重组表达载体,通过诱导表达获得InlG重组蛋白,将其纯化后免疫小鼠(100μg·只^(-1)),经细胞融合、克隆和筛选共获得了3株阳性杂交瘤细胞株,分别命名为1D2、1D2-1和2H10。经鉴定,其分泌抗体亚型均为IgG 1。利用制备的1D2-1作为捕获抗体,产单核细胞李氏杆菌兔多抗作为检测抗体,初步建立检测产单核细胞李氏杆菌的双抗体夹心ELISA方法。Western blot结果显示3株单克隆抗体(mAb)与InlG均可发生特异性反应。双抗体夹心ELISA方法的特异性试验结果显示,与沙门菌、大肠杆菌、枯草杆菌、白色葡萄球菌和柠檬色葡萄球菌均不发生反应。灵敏度试验结果显示,该方法检测LM纯培养物的检测限为1.0×10^(6)CFU·mL^(-1)。重复性试验结果显示,各组变异系数在5%~10%之间(<10%)。综上,本研究利用抗内化素G蛋白的mAb和兔多抗建立了双抗体夹心ELISA方法,该方法具有良好的特异性、敏感性和重复性,可用于产单核细胞李氏杆菌感染的快速诊断检测。This study aimed to establish a rapid method for the detection of Listeria monocytogenes.Listeria monocytogenes(LM)internalin G(InlG)gene was amplified by PCR,pET-32a-InlG recombinant expression vector was constructed,and recombinant protein of InlG was obtained by induced expression,which was purified and immunized to mice(100μg per mouse).Three positive hybridoma cell lines were obtained by cell fusion,cloning,and screening,and were designated as 1D2,1D2-1 and 2H10.The subtypes of the antibodies were identified as IgG 1.Using the prepared 1D2-1 as the capture antibody and the Listeria monocytogenes rabbit-derived polyclonal antibody as the detection antibody,a preliminary double-antibody sandwich ELISA method for detecting Listeria monocytogenes was established.WESTERN BLOT results showed that all three monoclonal antibodies(mAb)reacted specifically with InlG.The results of the specificity test of the method showed that there was not reaction to Salmonella,Escherichia coli,Bacillus subtilis,Staphylococcus albicans and Staphylococcus citreus.The results of the sensitivity test showed that the limit of the method for the detection of LM pure cultures was 1.0×10^(6) CFU·mL^(-1).The results of the reproducibility test showed that the coefficients of variation for each group ranged from 5%-10%(<10%).In conclusion,this study established a double antibody sandwich ELISA method using anti-internalin G protein mAb and rabbit polyclonal antibody.The method showed good specificity,sensitivity and repeatability,and can be used for rapid diagnosis and detection of Listeria monocytogenes infection.

关 键 词：产单核细胞李氏杆菌 内化素G 单克隆抗体 酶联免疫吸附试验 

分 类 号：S852.61[农业科学—基础兽医学] S854.43[农业科学—兽医学]