牛冠状病毒S1蛋白的真核表达及间接ELISA方法的建立与应用

doi:10.11843/j.issn.0366-6964.2024.05.023

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (5): 2050-2060.doi: 10.11843/j.issn.0366-6964.2024.05.023

牛冠状病毒S1蛋白的真核表达及间接ELISA方法的建立与应用

黄金^1,2, 李思远^1,6, 毛立^1,2, 蔡旭航^1,6, 谢玲玲⁴, 王府⁴, 周华⁵, 李基棕^1,2,3*, 李彬^1,2,3*

1. 江苏省农业科学院兽医研究所, 农业农村部兽用生物制品工程技术重点实验室, 南京 210014;
2. 南京农业大学动物医学院, 南京 210095;
3. 江苏大学生命科学学院/食品与生物工程学院, 镇江 212013;
4. 贵州省种畜禽种质测定中心, 贵阳 550018;
5. 黔西市动物疫病预防控制中心, 黔西 551500;
6. 西北农林科技大学动物医学院, 杨凌 712100

收稿日期:2023-07-10 出版日期:2024-05-23 发布日期:2024-05-27
通讯作者: 李基棕，主要从事动物传染病防治和诊断技术研究，E-mail:lijizong22@sina.com；李彬，主要从事动物病毒学方面研究，E-mail:libinana@126.com
作者简介:黄金(1999-)，女，江西赣州人，硕士生，主要从事动物传染病防治和诊断技术研究，E-mail:1748916481@qq.com；Tel:025-84390988
基金资助:
“十四五”国家重点研发计划(2021YFD1801101)；贵州省种业发展项目(肉牛遗传改良与高效养殖技术研究)

Eukaryotic Expression of Bovine Coronavirus S1 Protein and Establishment and Application of Indirect ELISA

HUANG Jin^1,2, LI Siyuan^1,6, MAO Li^1,2, CAI Xuhang^1,6, XIE Lingling⁴, WANG Fu⁴, ZHOU Hua⁵, LI Jizong^1,2,3*, LI Bin^1,2,3*

1. Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture and Rural Affairs, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
3. School of Food and Biological Engineering/School of Life Sciences, Jiangsu University, Zhenjiang 212013, China;
4. Guizhou Testing Center for Livestock and Poultry Germplasm, Guiyang 550018, China;
5. Qianxi Animal Disease Control Center, Qianxi 551500, China;
6. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China

Received:2023-07-10 Online:2024-05-23 Published:2024-05-27

摘要/Abstract

摘要： 为建立一种检测牛冠状病毒(BCoV)的抗体间接ELISA检测方法，本研究利用昆虫杆状病毒表达系统表达BCoV的S1蛋白，并作为包被抗原，建立BCoV的S1蛋白抗体间接ELISA检测方法，应用于临床样品检测。结果显示，S1蛋白大小约100 ku，可表达于昆虫High5细胞培养基上清，经Western blot鉴定S1蛋白抗原性良好；经反应条件优化，确定抗原包被浓度为0.125 μg·mL^-1，血清稀释度为1∶200；采用1%明胶，于37 ℃封闭3 h；血清样品于37 ℃孵育30 min；酶标二抗的稀释度1∶25 000，37 ℃孵育45 min；37 ℃避光显色13 min；临界值为0.297；经检验，该方法特异性良好；批间及批内变异系数均小于10％。当临界值为OD₄₅₀ _nm为0.297时，若以IFA为标准，ELISA的敏感性为96.88%(31/32)，特异性为87.50%(7/8)，与IFA具有很强的一致性(κ=0.844，P<0.01)；若以中和试验为标准，ELISA的敏感性为100.00%(31/31)，特异性为88.89%(8/9)，与血清中和试验具有很强的一致性(κ=0.925，P<0.01)。采用该方法对303份牛血清进行检测，BCoV阳性率为74.91%。由此说明，本研究建立的ELISA方法特异性强，敏感性高，重复性好，可用于BCoV的血清学调查和临床诊断，并可以应用于替代中和试验检测BCoV免疫后抗体水平。

关键词: 牛冠状病毒, 昆虫杆状病毒表达系统, S1蛋白, 间接ELISA

Abstract: In order to establish a specific indirect ELISA method that can replace the detection of neutralizing antibodies against bovine coronavirus (BCoV), the recombinant S1 protein of BCoV was prepared by expression of insect baculovirus expression system. The purified recombinant S1 protein was used as coating antigen to establish an indirect ELISA method for detection of BCoV S1 protein antibody. The results showed that the recombinant S1 protein was about 100 ku and could be expressed in the supernatant of insect High5 cell culture medium, and the antigenicity of S1 protein was identified by Western blot. After the reaction conditions were optimized, the antigen coating concentration was 0.125 μg·mL^-1, and the dilution of serum was 1∶200. The type of sealant is 1% gelatin and the sealing time is 3 h at 37 ℃. The action time of serum is incubating 30 min at 37 ℃. The dilution of enzyme-labeled second antibody was 1∶25 000, and the time was incubated at 37 ℃ for 45 min. The time for substrate coloration was 13 min at 37 ℃. The critical value was 0.297. This method had a good specificity, the maximum coefficients of variation of inter-assay and intra-assay repeatability tests were less than 10%. Taking IFA as the standard, the results showed that when the critical value OD_{450 nm} was 0.297, the sensitivity of IFA was 96.88% (31/32) and the specificity was 87.50% (7/8), which was highly consistent with the serum neutralization test (κ=0.844, P<0.01). Taking the neutralization test as the standard, the results showed that when the critical value OD_{450 nm} was 0.297, the sensitivity of ELISA was 100.00% (31/31) and the specificity was 88.89% (8/9), which was highly consistent with the serum neutralization test (κ=0.925, P<0.01). Three hundred and three samples of bovine serum were detected by this method, and the positive rate was 74.91%. Therefore, the established ELISA method has strong specificity, high sensitivity and good repeatability, which can be used as an alternative to neutralization test to detect BCoV antibody levels after immunization.

Key words: bovine coronavirus, insect baculovirus expression system, S1 protein, indirect ELISA

中图分类号:

S852.659.6

黄金, 李思远, 毛立, 蔡旭航, 谢玲玲, 王府, 周华, 李基棕, 李彬. 牛冠状病毒S1蛋白的真核表达及间接ELISA方法的建立与应用[J]. 畜牧兽医学报, 2024, 55(5): 2050-2060.

HUANG Jin, LI Siyuan, MAO Li, CAI Xuhang, XIE Lingling, WANG Fu, ZHOU Hua, LI Jizong, LI Bin. Eukaryotic Expression of Bovine Coronavirus S1 Protein and Establishment and Application of Indirect ELISA[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2050-2060.

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牛冠状病毒S1蛋白的真核表达及间接ELISA方法的建立与应用

Eukaryotic Expression of Bovine Coronavirus S1 Protein and Establishment and Application of Indirect ELISA

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