鸡痘病毒ORF127蛋白间接ELISA抗体检测方法的建立与应用

doi:10.3969/j.issn.1004-1524.20250139

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (10): 2057-2065.DOI: 10.3969/j.issn.1004-1524.20250139

鸡痘病毒ORF127蛋白间接ELISA抗体检测方法的建立与应用

华炯钢¹(), 朱寅初¹, 叶加林², 张存¹, 陈柳¹, 倪征¹, 付媛¹, 霍苏馨¹, 云涛¹^,^*()

1.浙江省农业科学院畜牧兽医研究所,全省畜禽生物育种重点实验室,农业农村部畜禽资源(家禽)评价利用重点实验室,家禽种业与绿色养殖技术浙江省工程研究中心,浙江杭州 310021
2.杭州高级中学国际部,浙江杭州 310020

收稿日期:2025-02-24 出版日期:2025-10-25 发布日期:2025-11-13
作者简介:华炯钢(1968—),男,浙江杭州人,副研究员,主要从事家禽疫病防控研究。E-mail:569102400@qq.com
通讯作者: *云涛,E-mail:yunt116@zaas.ac.cn
基金资助:
浙江省“尖兵”“领雁”研发攻关计划(2023C02022);浙江省农业重大技术协同推广项目(2023ZDXT15);浙江省“三农九方”科技协作计划项目(2023SNJF042)

Establishment and application of an indirect ELISA method for detecting antibodies against fowlpox virus ORF127 protein

HUA Jionggang¹(), ZHU Yinchu¹, YE Jialin², ZHANG Cun¹, CHEN Liu¹, NI Zheng¹, FU Yuan¹, HUO Suxin¹, YUN Tao¹^,^*()

1. Zhejiang Key Laboratory of Livestock and Poultry Biotech Breeding, Key Laboratory of Livestock and Poultry Resources (Poultry) Evaluation and Utilization, Ministry of Agriculture and Rural Affairs, Zhejiang Engineering Research Center for Poultry Breeding Industry and Green Farming Technology, Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2. International Division, Hangzhou High School, Hangzhou 310020, China

Received:2025-02-24 Online:2025-10-25 Published:2025-11-13

摘要/Abstract

摘要： 为建立快速、准确的鸡痘病毒(fowlpox virus, FPV)抗体检测方法,将FPV ORF127基因的抗原表位区域克隆于pET-28a(+)原核表达载体,诱导表达并纯化获得FPV ORF127截短蛋白,以纯化的蛋白作为包被抗原,通过优化各反应条件,建立检测FPV抗体的间接ELISA方法,经特异性、敏感性、重复性等试验和临床血清样本检测进行系统验证。结果显示,间接ELISA检测FPV抗体的最佳反应条件为:包被抗原质量浓度为10 μg·mL^-1,待检血清稀释度为1∶100,酶标二抗稀释度为1∶25 000;特异性试验结果显示,除FPV阳性血清呈阳性反应外,与其他6种病毒(新城疫病毒、H9亚型禽流感病毒、鸡传染性法氏囊病毒、鸡传染性喉气管炎病毒、禽腺病毒4型、火鸡疱疹病毒)的阳性血清均无交叉反应;敏感性结果显示,FPV阳性血清稀释800倍后检测结果仍为阳性;批内、批间重复试验结果显示,批内变异系数为1.81%~7.07%,批间变异系数为2.12%~7.16%,均小于10%。临床血清样品检测结果显示,180份血清样本总阳性率为79.4%(143/180)。上述结果表明,本研究建立的间接ELISA方法特异性强、敏感性高、重复性和稳定性好,可为免疫鸡群FPV抗体水平监测和FPV流行病学调查提供技术支撑。

关键词: 鸡痘病毒, ORF127蛋白, 间接ELISA, 抗体

Abstract:

To establish a rapid and accurate method for detecting fowlpox virus (FPV) antibodies, this study cloned the antigenic epitope region of FPV ORF127 gene into the pET-28a(+) prokaryotic expression vector. The truncated FPV ORF127 protein was successfully expressed and purified. Using this recombinant protein as a coating antigen, an indirect ELISA was developed through systematic optimization of reaction parameters. The method was comprehensively validated, including specificity, sensitivity, repeatability tests, and clinical serum sample detection. The results showed that the optimal reaction conditions for the indirect ELISA were as follows: coating antigen concentration of 10 μg·mL^-1, serum dilution of 1∶100, and enzyme-labeled secondary antibody dilution of 1∶25 000. Specificity tests revealed that except for FPV-positive serum, no cross-reactions were observed with positive sera against six other viruses (Newcastle disease virus, H9 subtype avian influenza virus, infectious bursal disease virus, infectious laryngotracheitis virus, fowl adenovirus type 4, and turkey herpesvirus). Sensitivity tests showed that FPV-positive serum remained detectable at a dilution of 1∶800. Reproducibility tests demonstrated that the intra-assay and inter-assay coefficients of variation were 1.81%-7.07% and 2.12%-7.16%, respectively, both below 10%. Clinical serum sample testing showed a total positivity rate of 79.4% (143/180) among 180 samples. These results indicate that the established indirect ELISA method exhibits high specificity, sensitivity, reproducibility, and stability. It can provides a reliable tool for detecting FPV antibodies in vaccinated flocks and supporting FPV epidemiological investigations.

Key words: fowlpox virus, ORF127 protein, indirect ELISA, antibody

中图分类号:

S852.657

华炯钢, 朱寅初, 叶加林, 张存, 陈柳, 倪征, 付媛, 霍苏馨, 云涛. 鸡痘病毒ORF127蛋白间接ELISA抗体检测方法的建立与应用[J]. 浙江农业学报, 2025, 37(10): 2057-2065.

HUA Jionggang, ZHU Yinchu, YE Jialin, ZHANG Cun, CHEN Liu, NI Zheng, FU Yuan, HUO Suxin, YUN Tao. Establishment and application of an indirect ELISA method for detecting antibodies against fowlpox virus ORF127 protein[J]. Acta Agriculturae Zhejiangensis, 2025, 37(10): 2057-2065.

图/表 8

图1 FPV ORF127蛋白抗原表位分析结果框内为FPV ORF127截短蛋白区。

Fig.1 Analysis result of antigenic epitopes of FPV ORF127 protein The truncated protein region of FPV ORF127 was in the frame.

图2 重组质粒PCR(A)和酶切(B)鉴定结果 M1,DL 2 000 DNA marker;M2,DL 15 000 DNA marker;1,pET-28-FPV-127 PCR产物;2,阴性对照;3,pET-28。

Fig.2 PCR (A) and restriction enzyme digestion (B) identification results of the recombinant plasmid M1, DL 2 000 DNA marker; M2, DL 15 000 DNA marker; 1, PCR products of pET-28-FPV-127; 2, Negative control; 3, pET-28.

图3 FPV-127截短蛋白的SDS-PAGE电泳(A)和Western blot鉴定(B) M,预染蛋白分子量标准(10~180 ku);1,未诱导的pET-28a-127/BL21;2,诱导的pET-28a-127/BL21;3,裂解后上清液;4,裂解后沉淀;5,镍柱纯化后的FPV-127截短蛋白。

Fig.3 SDS-PAGE electrophoresis (A) and Western blot identification (B) of FPV-127 truncated protein M, Color prestained protein marker(10-180 ku); 1, Uninduced pET-28a-127/BL21; 2, Induced pET-28a-127/BL21; 3, Supernatant after cell lysis; 4, Precipitation after cell lysis; 5, FPV-127 truncated protein purified by nickel affinity chromatography.

表1 不同质量浓度的抗原与不同稀释度的血清在450 nm的吸光度

Table 1 Absorbance at 450 nm of antigens with different mass concentrations and serum with different dilutions

血清稀释度 Serum dilution	项目 Item	不同FPV-127抗原质量浓度的吸光度Absorbance of FPV-127 antigens with different mass concentration
血清稀释度 Serum dilution	项目 Item	20 μg·mL^-1	10 μg·mL^-1	5 μg·mL^-1	2.5 μg·mL^-1	1.25 μg·mL^-1	0.625 μg·mL^-1
1∶25	P	2.220	2.003	1.590	1.138	0.793	0.671
	N	0.673	0.403	0.243	0.167	0.129	0.210
	P/N	3.299	4.970	6.543	6.814	6.117	3.195
1∶50	P	2.146	1.799	1.130	0.753	0.486	0.404
	N	0.389	0.243	0.145	0.101	0.088	0.086
	P/N	5.517	7.403	7.793	7.485	5.523	4.698
1∶100	P	2.117	1.474	0.896	0.497	0.291	0.252
	N	0.257	0.148	0.096	0.074	0.062	0.066
	P/N	8.237	9.959	9.333	6.716	4.694	3.818
1∶200	P	1.896	1.145	0.562	0.311	0.189	0.149
	N	0.215	0.123	0.099	0.065	0.067	0.054
	P/N	8.817	9.309	5.677	4.785	2.821	2.759

表2 血清稀释100倍时不同稀释度酶标二抗在450 nm的吸光度

Table 2 Absorbance at 450 nm of enzyme-labeled secondary antibody with different dilutions when serum was diluted 100-fold

项目 Item	不同酶标二抗稀释度的吸光度Absorbance with different dilutions of enzyme-labeled second antibody
项目 Item	1∶10 000	1∶15 000	1∶20 000	1∶25 000	1∶30 000	1∶35 000
P	2.227	2.073	1.833	1.693	1.540	1.352
N	0.207	0.133	0.110	0.108	0.103	0.101
P/N	10.758	15.586	16.664	15.676	14.951	13.386

表3 间接ELISA方法的敏感性试验结果

Table 3 Sensitivity test results of indirect ELISA method

血清 Serum	稀释倍数 Dilution ratio	D₄₅₀ D₄₅₀
阳性血清Positive serum	100	1.394
	200	0.988
	400	0.652
	800	0.403
	1 600	0.259
	3 200	0.165
	6 400	0.117
阴性血清Negative serum	100	0.123

表4 间接ELISA重复性试验结果

Table 4 Repeatability test results of indirect ELISA method

血清编号 Serum number	批内重复试验Intra batch repeated experiment			批间重复试验Inter batch repeated experiment
血清编号 Serum number	D₄₅₀平均值 Average of D₄₅₀ value	标准差 Standard deviation	变异系数 Coefficient of variation/%	D₄₅₀平均值 Average of D₄₅₀ value	标准差 Standard deviation	变异系数 Coefficient of variation/%
1	1.448	0.029	2.00	1.410	0.041	2.91
2	1.161	0.021	1.81	1.132	0.024	2.12
3	0.779	0.015	1.93	0.768	0.022	2.86
4	0.615	0.019	3.08	0.628	0.045	7.16
5	0.388	0.012	3.09	0.376	0.015	3.99
6	0.341	0.014	4.09	0.352	0.012	3.41
7	0.163	0.009	5.52	0.162	0.010	6.17
8	0.113	0.008	7.07	0.110	0.004	3.63

图4 临床样品的间接ELISA方法检测结果 NC,FPV阴性血清样品;1~6,各鸡场FPV疫苗免疫后采集的血清样品。

Fig.4 Indirect ELISA detection results of clinical samples NC, FPV negative serum samples; 1-6, Serum samples collected from each chicken farm after FPV vaccine immunization.

参考文献 23

[1]	BERTELLONI F, CECCHERELLI R, MARZONI M, et al. Molecular detection of Avipox virus in wild birds in central Italy[J]. Animals, 2022, 12(3): 338.
[2]	MATOS M, BILIC I, PALMIERI N, et al. Epidemic of cutaneous fowlpox in a naïve population of chickens and turkeys in Austria: detailed phylogenetic analysis indicates co-evolution of fowlpox virus with reticuloendotheliosis virus[J]. Transboundary and Emerging Diseases, 2022, 69(5): 2913-2923.
[3]	姜绍全, 王金亮, 秦燕. 海兰褐蛋鸡痘病毒的分离与鉴定[J]. 山东畜牧兽医, 2015, 36(1): 36-37.
	JIANG S Q, WANG J L, QIN Y. Isolation and identification of poxvirus from hailan brown laying hens[J]. Shandong Journal of Animal Science and Veterinary Medicine, 2015, 36(1): 36-37. (in Chinese)
[4]	KIM T J, SCHNITZLEIN W M, MCALOOSE D, et al. Characterization of an avianpox virus isolated from an Andean condor (Vultur gryphus)[J]. Veterinary Microbiology, 2003, 96(3): 237-246.
[5]	YEHIA N, ELSAYED S, AL-SAEED F A, et al. Current situation and genomic characterization of fowlpox virus in lower Egypt during 2022[J]. Poultry Science, 2023, 102(8): 102769.
[6]	AFONSO C L, TULMAN E R, LU Z, et al. The genome of fowlpox virus[J]. Journal of Virology, 2000, 74(8): 3815-3831.
[7]	WELI S C, TRYLAND M. Avipoxviruses: infection biology and their use as vaccine vectors[J]. Virology Journal, 2011, 8: 49.
[8]	赵宏吉, 张金花, 司朵朵, 等. 山羊痘病毒G9蛋白的原核表达及间接ELISA方法的建立[J]. 中国预防兽医学报, 2021, 43(3): 280-284.
	ZHAO H J, ZHANG J H, SI D D, et al. Prokaryotic expression of G9 protein of GTPV and establishment of an indirect ELISA[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(3): 280-284. (in Chinese with English abstract)
[9]	叶伟成, 云涛, 朱寅初, 等. 基于番鸭细小病毒VP3蛋白的间接ELISA方法建立[J]. 浙江农业科学, 2022, 63(11): 2740-2745, 2750.
	YE W C, YUN T, ZHU Y C, et al. Establishment of indirect ELISA assay based on VP3 protein of Muscovy duck parvovirus[J]. Journal of Zhejiang Agricultural Sciences, 2022, 63(11): 2740-2745, 2750. (in Chinese with English abstract)
[10]	OJEDA S, DOMI A, MOSS B. Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex[J]. Journal of Virology, 2006, 80(19): 9822-9830.
[11]	GRAY R D M, ALBRECHT D, BEERLI C, et al. Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion[J]. Nature Microbiology, 2019, 4(10): 1636-1644.
[12]	FOO C H, WHITBECK J C, PONCE-DE-LEÓN M, et al. The myristate moiety and amino terminus of vaccinia virus l1 constitute a bipartite functional region needed for entry[J]. Journal of Virology, 2012, 86(10): 5437-5451.
[13]	SHINODA K, WYATT L S, MOSS B. The neutralizing antibody response to the vaccinia virus A28 protein is specifically enhanced by its association with the H2 protein[J]. Virology, 2010, 405(1): 41-49.
[14]	ADEBAJO M C, ADEMOLA S I, OLUWASEUN A. Seroprevalence of fowl pox antibody in indigenous chickens in jos north and south council areas of Plateau state, Nigeria: implication for vector vaccine[J]. International Scholarly Research Notices, 2012, 2012(1): 154971.
[15]	MOCKETT A P A, SOUTHEE D J, TOMLEY F M, et al. Fowlpox virus: its structural proteins and immunogens and the detection of viral-specific antibodies by ELISA[J]. Avian Pathology, 1987, 16(3): 493-504.
[16]	雪鹏, 刘悦竹, 姜世金, 等. 用间接ELISA检测鸡痘病毒抗体的建立与应用[J]. 中国兽医杂志, 2005, 41(6): 15-16.
	WANG X P, LIU Y Z, JIANG S J, et al. Establishment and application of an indirect ELISA to detect antibodies against poultry pox[J]. Chinese Journal of Veterinary Medicine, 2005, 41(6): 15-16. (in Chinese)
[17]	张体银, 孙蕾, 孙学辉, 等. 应用间接ELISA检测鸡痘病毒抗体方法的建立[J]. 中国预防兽医学报, 2005, 27(6): 540-543.
	ZHANG T Y, SUN L, SUN X H, et al. Development of indirect ELISA for detection of antibodies against fowlpox virus[J]. Chinese Journal of Preventive Veterinary Medicine, 2005, 27(6): 540-543. (in Chinese with English abstract)
[18]	纪朋艳, 卢广林. 检测鸡痘病毒抗体间接ELISA方法的建立[J]. 黑龙江畜牧兽医, 2012(23): 94-95.
	JI P Y, LU G L. Establishment of indirect ELISA for detection of fowlpox virus antibody[J]. Heilongjiang Animal Science and Veterinary Medicine, 2012(23): 94-95. (in Chinese)
[19]	张琪, 庞文静, 付明哲, 等. 山羊痘病毒ORF103蛋白的原核表达及间接ELISA抗体检测方法的建立与应用[J]. 中国兽医科学, 2017, 47(5): 537-543.
	ZHANG Q, PANG W J, FU M Z, et al. Prokaryotic expression of ORF103 protein of capripox virus and establishment of indirect ELISA for the detection of antibodies against the virus[J]. Chinese Veterinary Science, 2017, 47(5): 537-543. (in Chinese with English abstract)
[20]	VENKATESAN G, KUMAR TELI M, SANKAR M, et al. Expression and evaluation of recombinant P32 protein based ELISA for sero-diagnostic potential of capripox in sheep and goats[J]. Molecular and Cellular Probes, 2018, 37: 48-54.
[21]	DASHPRAKASH M, VENKATESAN G, KUMAR A, et al. Prokaryotic expression, purification and evaluation of goatpox virus ORF117 protein as a diagnostic antigen in indirect ELISA to detect goatpox[J]. Archives of Virology, 2019, 164(4): 1049-1058.
[22]	邓舜洲, 蒋新华, 冷闯, 等. 猪痘病毒P35基因的原核表达及间接ELISA抗体检测方法的建立[J]. 中国兽医学报, 2013, 33(10): 1488-1492.
	DENG S Z, JIANG X H, LENG C, et al. Establishment of an indirect ELISA for detection of antibodies against swinepox virus with the recombinant P35 as the coating antigen[J]. Chinese Journal of Veterinary Science, 2013, 33(10): 1488-1492. (in Chinese with English abstract)
[23]	朱俊达, 王爽, 任书凝, 等. 基于猴痘病毒A27L蛋白的间接ELISA抗体检测方法的建立[J]. 中国兽医杂志, 2022, 58(10): 1-7.
	ZHU J D, WANG S, REN S N, et al. Establishment of indirect ELISA method for detection of monkeypox virus antibody based on A27L protein[J]. Chinese Journal of Veterinary Medicine, 2022, 58(10): 1-7. (in Chinese with English abstract)

鸡痘病毒ORF127蛋白间接ELISA抗体检测方法的建立与应用

Establishment and application of an indirect ELISA method for detecting antibodies against fowlpox virus ORF127 protein

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 23

相关文章 15

编辑推荐

Metrics

本文评价

[1]	沈峥嵘, 戴远兴, 郭留明, 汪芷瑶, 张恒木. 中国小麦花叶病毒(CWMV)外壳蛋白(CP)特异性抗体的制备与应用[J]. 浙江农业学报, 2024, 36(9): 2042-2050.
[2]	李菊, 毕冬琳, 杨晓莉, 杨东亮, 张潇文, 刘方程, 李琼毅, 柏家林. 小反刍兽疫病毒非结构蛋白C单克隆抗体的制备与鉴定[J]. 浙江农业学报, 2024, 36(5): 1047-1054.
[3]	李天恩, 周思含, 孙洪超, 付媛, 石团员, 闫文朝. 鸡柔嫩艾美耳球虫2种假定致密颗粒蛋白基因的克隆与表达[J]. 浙江农业学报, 2024, 36(3): 503-514.
[4]	薛姣雄, 赵婷芳, 张倩, 唐青海, 高翠翠, 赵铖, 张妍, 全飞杨, 刘婷, 杨灿, 杨海, 王文秀. 犬冠状病毒S1蛋白特异性小分子抗体Fab的制备[J]. 浙江农业学报, 2023, 35(11): 2568-2583.
[5]	唐国亮, 张玉宝, 王若愚, 王亚军, 赵霞, 苏学思, 金卫杰. 魔芋花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2022, 34(11): 2471-2481.
[6]	朱寅初, 王宏宇, 云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 浙江地区鹅星状病毒分离鉴定及其衣壳蛋白多克隆抗体的制备[J]. 浙江农业学报, 2022, 34(10): 2149-2159.
[7]	沈卫锋, 郭琦, 刘莉, 牛宝龙, 翁宏飚, 楼宝. 虾肝肠胞虫(Enterocytozoon hepatopenaei)SWP2基因的克隆、表达及其在虾类病害检测中的应用[J]. 浙江农业学报, 2021, 33(6): 993-1000.
[8]	刘君雯, 王迪, 朱艳艳, 邢刚, 占松鹤, 刘晓露, 魏建忠, 孙裴, 刘雪兰, 李郁. 猪丹毒丝菌CbpB基因的克隆表达及其间接ELISA抗体检测方法的建立与应用[J]. 浙江农业学报, 2021, 33(5): 816-824.
[9]	苏学思, 张玉宝, 王若愚, 王亚军, 唐国亮, 金卫杰. 车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 104-111.
[10]	徐海, 王健, 郭长明, 董洪燕, 邓碧华, 侯继波. 单域抗体T7噬菌体展示文库构建与鉴定[J]. 浙江农业学报, 2021, 33(1): 27-33.
[11]	刘金玉, 黄鹰. 粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 34-42.
[12]	潘传燕, 林勇, 冯鹏霏, 张永德, 罗洪林. 尼罗罗非鱼Hsc70的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2019, 31(8): 1272-1279.
[13]	张康, 张璇, 闫遵祥, 王磊, 张凯, 张景艳, 罗永江, 仇正英, 薛欢, 李建喜. 牛病毒性腹泻病毒Core蛋白的原核表达及多克隆抗体制备[J]. 浙江农业学报, 2019, 31(11): 1819-1824.
[14]	彭琪琪, 羊健, 廖乾生, 张恒木. 一个植物半胱氨酸蛋白酶多克隆抗体的制备及其应用[J]. 浙江农业学报, 2018, 30(6): 881-885.
[15]	延涵, 杨瑞秀, 盖晓彤, 李刚, 赵治国, 高增贵. 甜瓜枯萎病菌ELISA检测方法的初步建立[J]. 浙江农业学报, 2018, 30(12): 2081-2086.