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

doi:10.11843/j.issn.0366-6964.2024.09.031

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (9): 4069-4076.doi: 10.11843/j.issn.0366-6964.2024.09.031

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

马金锐¹(), 史文静¹, 田常青¹, 董志杰¹, 赵学慧¹, 芝吉¹, 曹青¹, 魏衍全¹, 宋维丽², 薛惠文¹, 苟惠天¹^,*()

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

收稿日期:2023-11-13 出版日期:2024-09-23 发布日期:2024-09-27
通讯作者: 苟惠天 E-mail:Mjr_2473@163.com;gouht@gsau.edu.cn
作者简介:马金锐(1996-), 男, 甘肃天水人, 硕士生, 主要从事兽医公共卫生相关研究, E-mail: Mjr_2473@163.com
基金资助:
国家自然科学基金项目(31960726);国家自然科学基金项目(32060822);国家自然科学基金项目(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)

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

Jinrui MA¹(), Wenjing SHI¹, Changqing TIAN¹, Zhijie DONG¹, Xuehui ZHAO¹, Ji ZHI¹, Qing CAO¹, Yanquan WEI¹, Weili SONG², Huiwen XUE¹, Huitian GOU¹^,*()

1. College of Veterinary Medicine Gansu Agricultural University, Lanzhou 730070, China
2. Animal Husbandry and Veterinary Station, Longmen Town, Lintao County, Lintao 730500, China

Received:2023-11-13 Online:2024-09-23 Published:2024-09-27
Contact: Huitian GOU E-mail:Mjr_2473@163.com;gouht@gsau.edu.cn

摘要/Abstract

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(100 μg·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IgG₁。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1.0×10⁶ 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。

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

Abstract:

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 (100 μg 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 IgG₁. 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 1.0×10⁶ 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.

Key words: Listeria monocytogenes, internalin G, monoclonal antibody, enzyme-linked immunosorbent assay

中图分类号:

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, 苟惠天. 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[J]. 畜牧兽医学报, 2024, 55(9): 4069-4076.

Jinrui MA, Wenjing SHI, Changqing TIAN, Zhijie DONG, Xuehui ZHAO, Ji ZHI, Qing CAO, Yanquan WEI, Weili SONG, Huiwen XUE, Huitian GOU. Preparation of Monoclonal Antibodies against Internalin G of Listeria monocytogenes and Their Preliminary Application[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4069-4076.

图/表 5

图 1

图 2

图 3

表 1

表 2

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试验菌株Experimental strain	ELISA反应结果ELISA Result	试验菌株Experimental strain	ELISA反应结果ELISA Result
产单核细胞李氏杆菌Listeria monocytogenes	+	沙门菌Salmonella	-
伊氏李斯特菌Listeria ivanovii	+	大肠杆菌Escherichia coli	-
韦氏李斯特菌Listeria welshimeri	+	枯草杆菌Bacillus subtilis	-
无害李斯特菌Listeria innocua	+	白色葡萄球菌Staphylococcus albus	-
		柠檬色葡萄球菌Staphylococcus citreus	-

样品增菌液Enrichment solution of samples	接菌浓度/(CFU·mL^-1) Concentration of inoculum	增菌时间/h Enrichment of time
样品增菌液Enrichment solution of samples	接菌浓度/(CFU·mL^-1) Concentration of inoculum	12	15	18	21	24
生牛乳Raw milk	1.0×10⁰	0.572	0.796	0.932	0.942	1.582
	1.0×10¹	0.751	0.880	0.938	1.040	1.740
	1.0×10²	0.827	0.899	1.097	2.096	3.267
	1.0×10³	0.831	1.354	1.834	2.385	3.669
	1.0×10⁴	0.932	1.688	2.583	2.523	4.636
	1.0×10⁵	1.366	2.350	2.946	2.846	5.210
	1.0×10⁶	2.767	3.065	2.980	4.546	5.504
生牛肉Raw beef	1.0×10⁰	0.825	0.876	1.001	1.746	1.805
	1.0×10¹	0.896	0.997	1.048	1.984	1.997
	1.0×10²	1.010	1.107	1.944	2.256	2.262
	1.0×10³	1.081	1.509	1.903	2.365	2.591
	1.0×10⁴	1.441	1.747	2.126	2.380	2.631
	1.0×10⁵	2.062	3.677	2.477	2.589	2.777
	1.0×10⁶	2.169	2.381	2.902	3.006	3.939

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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

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