羊痘病毒抗体胶体金免疫层析试纸条的研制与初步应用

doi:10.11843/j.issn.0366-6964.2025.07.029

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (7): 3368-3377.doi: 10.11843/j.issn.0366-6964.2025.07.029

羊痘病毒抗体胶体金免疫层析试纸条的研制与初步应用

何印娣¹^,²(), 石正旺², 石鑫泰², 陈婕², 廖焕程², 张帆², 罗俊聪², 朱昱茜¹^,², 席韬², 李帅鹏¹^,², 王川¹^,*(), 田宏²^,*(), 郑海学²^,*()

1. 甘肃农业大学动物医学院, 兰州 730070
2. 中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室, 兰州 730000

收稿日期:2024-09-23 出版日期:2025-07-23 发布日期:2025-07-25
通讯作者: 王川,田宏,郑海学 E-mail:2498727818@qq.com;wangchuan@gsau.edu.cn;xibeitian0931@163.com;zhenghaixue@caas.cn
作者简介:何印娣(1998-)，女，甘肃临夏人，硕士生，主要从事动物疫病诊断技术研究，E-mail：2498727818@qq.com
基金资助:
甘肃省科技重大专项课题(22ZD6NA001)；中央高校基本科研业务费专项资金资助(学科交叉创新团队建设项目)(lzujbky-2022-ct02)；农业部科技创新2030-重大项目(2023ZD0404301)；中国农业科学院创新计划(CAAS-CSLPDCP-2023002)；“十四五”广东省揭榜挂帅项目(2023SDZG02)；2022甘肃省创新联合体项目(22ZD6NA012)

Development and Preliminary Application of Colloidal Gold Immunochromatographic Test Strips for Antibodies against Capripoxvirus

HE Yindi¹^,²(), SHI Zhengwang², SHI Xintai², CHEN Jie², LIAO Huancheng², ZHANG Fan², LUO Juncong², ZHU Yuqian¹^,², XI Tao², LI Shuaipeng¹^,², WANG Chuan¹^,*(), TIAN Hong²^,*(), ZHENG Haixue²^,*()

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute of Chinese Academy of Agriculture Sciences, Lanzhou 730000, China

Received:2024-09-23 Online:2025-07-23 Published:2025-07-25
Contact: WANG Chuan, TIAN Hong, ZHENG Haixue E-mail:2498727818@qq.com;wangchuan@gsau.edu.cn;xibeitian0931@163.com;zhenghaixue@caas.cn

摘要/Abstract

摘要：

旨在建立一种快速、简便、特异、灵敏的羊痘病毒抗体的通用型胶体金检测方法。通过原核表达、纯化获得羊痘病毒122重组蛋白，并制备122蛋白多克隆抗体。将122蛋白与胶体金偶联，作为金标抗原，再将122蛋白及兔多克隆抗体包被在硝酸纤维素膜(NC)上，分别作为检测线(T线)和质控线(C线)，经条件优化，研制成检测羊痘病毒抗体的胶体金免疫层析试纸条。结果表明，成功获得122重组蛋白，大小约为32 ku，胶体金试纸条可在12 min特异性检测到羊痘病毒抗体；与其他常见家畜疫病病原阳性血清无交叉反应；检测绵羊痘病毒、牛结节性皮肤病病毒和山羊痘病毒阳性血清的灵敏度分别为1 ∶64、1 ∶128和1 ∶128，与市售羊痘抗体间接ELISA诊断试剂盒(效价1 ∶128、1 ∶256和1 ∶256)的敏感性相当；对130份临床血清样品的检测与商品化试剂盒检测结果进行比较，两者的kappa值为0.93，为高度一致。本研究成功研制了羊痘病毒抗体通用型胶体金免疫层析试纸条，具有较高的敏感性和特异性，且具有成本低、检测快速、操作简便、结果容易判断等特点，对羊痘现场检测具有实际应用价值。

关键词: 羊痘病毒, 122蛋白, 多克隆抗体, 胶体金试纸条

Abstract:

This study aimed to establish a rapid, simple, specific and sensitive colloidal gold-based universal detection method for antibodies against Capripoxvirus. By constructing a recombinant vector and expressing and purifying the recombinant 122 protein of the Capripoxvirus in a prokaryotic system, rabbit polyclonal antibodies were prepared. The 122 protein was conjugated with colloidal gold as the gold-labeled antigen. Then, the 122 protein and rabbit polyclonal antibodies were coated onto a nitrocellulose membrane (NC) as the test line (T line) and control line (C line), respectively. After optimizing the conditions, a colloidal gold immunochromatographic test strip for detecting Capripoxvirus antibodies was developed. The results showed that the prokaryotically expressed recombinant 122 protein was approximately 32 ku in size. The developed colloidal gold test strip could specifically detect Capripoxvirus antibodies within 12 minutes; there was no cross-reaction with positive sera of other common livestock diseases. The sensitivity for detecting sheeppox virus lumpy skin disease virus and goat-pox virus positive sera was 1 ∶64, 1 ∶128, and 1 ∶128, respectively, which was comparable to the sensitivity of the commercially available indirect ELISA diagnostic kit for Capripoxvirus antibodies (titers 1 ∶128, 1 ∶256, 1 ∶256). The detection of 130 clinical serum samples was compared with the results of the commercial kit, and the kappa value was 0.93, indicating a high degree of consistency. This study successfully developed a universal colloidal gold immunochromatographic test strip for detecting Capripoxvirus antibodies, which has high sensitivity and specificity, as well as low cost, rapid detection, simple operation, and easy interpretation of results, providing practical application value for on-site detection of Capripoxvirus.

Key words: Capripoxvirus, protein 122, polyclonal antibodies, colloidal gold test strips

中图分类号:

S852.659.1

何印娣, 石正旺, 石鑫泰, 陈婕, 廖焕程, 张帆, 罗俊聪, 朱昱茜, 席韬, 李帅鹏, 王川, 田宏, 郑海学. 羊痘病毒抗体胶体金免疫层析试纸条的研制与初步应用[J]. 畜牧兽医学报, 2025, 56(7): 3368-3377.

HE Yindi, SHI Zhengwang, SHI Xintai, CHEN Jie, LIAO Huancheng, ZHANG Fan, LUO Juncong, ZHU Yuqian, XI Tao, LI Shuaipeng, WANG Chuan, TIAN Hong, ZHENG Haixue. Development and Preliminary Application of Colloidal Gold Immunochromatographic Test Strips for Antibodies against Capripoxvirus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(7): 3368-3377.

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储存时间/月Storage time(month)	室温(18~25 ℃)Room temperature(18~25 ℃)	4 ℃环境4 ℃ environment
0	+	+
1	+	+
3	+	+
6	+	+
9	-	+
12	-	+
15	-	-

方法Method		Biovetest ELISA试剂盒Biovetest ELISA kit
方法Method		阳性Positive	阴性Negative	合计Total
试纸条Test strip	阳性Positive	41	0	41
	阴性Negative	4	85	89
	合计Total	45	85	130
Kappa值Kappa value	0.93
Kappa value

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羊痘病毒抗体胶体金免疫层析试纸条的研制与初步应用

Development and Preliminary Application of Colloidal Gold Immunochromatographic Test Strips for Antibodies against Capripoxvirus

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