基于非洲猪瘟病毒p30与p54蛋白表位串联多肽的间接ELISA抗体检测方法的建立

doi:10.11843/j.issn.0366-6964.2022.12.018

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (12): 4325-4336.doi: 10.11843/j.issn.0366-6964.2022.12.018

基于非洲猪瘟病毒p30与p54蛋白表位串联多肽的间接ELISA抗体检测方法的建立

马俊^1,2, 王志远^1,2, 梁杏玲^1,2, 郑泽中^1,2, 杨汉春³, 张桂红^1,2,4, 王衡^1,2,5*

1. 华南农业大学兽医学院广东省临床重大疾病综合防控重点实验室, 广州 510642;
2. 国家非洲猪瘟区域实验室(广州), 广州 510642;
3. 中国农业大学动物医学院, 北京 100193;
4. 岭南现代农业科学与技术广东省实验室茂名分中心, 茂名 525000;
5. 华南农业大学非洲猪瘟防控技术研究中心与国家生猪种业工程技术研究中心, 广州 510642

收稿日期:2022-06-21 出版日期:2022-12-23 发布日期:2022-12-25
通讯作者: 王衡，主要从事兽医病理学研究，E-mail:wangheng2009@scau.edu.cn
作者简介:马俊(1996-)，女，河南信阳人，硕士生，主要从事兽医病理学研究，E-mail:junma@stu.scau.edu.cn
基金资助:
国家自然科学基金专项项目(31941004)；广州市重点领域研发计划(202206010036)；财政部和农业农村部(国家现代农业产业技术体系)

Development of an Indirect ELISA Antibodies Detection Method on Tandem-epitope Peptide of African Swine Fever Virus p30 and p54 Proteins

MA Jun^1,2, WANG Zhiyuan^1,2, LIANG Xingling^1,2, ZHENG Zezhong^1,2, YANG Hanchun³, ZHANG Guihong^1,2,4, WANG Heng^1,2,5*

1. Key Laboratory of Comprehensive Prevention and Control for Severe Clinical Animal Diseases of Guangdong Province, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;
2. African Swine Fever Regional Laboratory of China (Guangzhou), Guangzhou 510642, China;
3. College of Veterinary Medicine, China Agricultural University, Beijing 100193, China;
4. Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming 525000, China;
5. Research Center for African Swine Fever Prevention and Control of South China Agricultural University & National Engineering Research Center for Breeding Swine Industry, Guangzhou 510642, China

Received:2022-06-21 Online:2022-12-23 Published:2022-12-25

摘要/Abstract

摘要： 旨在建立检测非洲猪瘟病毒(ASFV)血清抗体的间接ELISA方法。本研究以两株纯化的ASFV p30与p54蛋白单克隆抗体(mAbs)为靶分子，利用噬菌体展示十二肽库进行四轮生物淘选，筛选多肽表位，以氨基酸GGG为接头设计合成表位串联多肽作为包被抗原，通过棋盘滴定法确定间接ELISA的最佳反应条件，利用不同类型血清样本对建立的方法进行特异性分析、敏感度分析、稳定性分析及符合性评价。噬菌体淘选试验结果表明¹⁴⁶PAEPYTT¹⁵²为本实验室保存的mAb所识别的p54蛋白抗原表位核心序列。ELISA条件优化试验结果显示，以鸡卵白蛋白(OVA)作为N端偶联物的表位串联多肽抗原具有较低的非特异性血清反应背景，当多肽以碳酸盐缓冲液包被(2 μg·mL^-1)，血清以封闭液(1%明胶溶液)稀释100倍，辣根过氧化物酶(HRP)标记抗体以0.05% PBST溶液稀释5 000倍时，反应效果最佳；以上述优化后的条件确定了血清抗体阳性临界值为0.339。方法评价试验结果显示，该方法与经典猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)及猪伪狂犬病病毒(PRV)的抗体阳性血清均无交叉反应，能检测低至1 600倍稀释的ASFV阳性血清，具有较好的重复性。用该方法与商品化的ASFV抗体检测试剂盒同时检测320份猪血清样本，两种方法的相对特异性和相对敏感性分别为97.6%与97.3%，总体符合率达97.5%(312/320)。综上表明，本研究建立的多肽间接ELISA方法具有良好的特异性、敏感性及重复性，具有发展为临床诊断试剂盒的潜在应用价值。

关键词: 非洲猪瘟病毒, p30蛋白, p54蛋白, 生物淘选, 表位串联多肽, 间接ELISA

Abstract: Here, we report the development of an indirect ELISA antibodies detection method for African swine fever virus (ASFV). Two purified monoclonal antibodies (mAbs) against ASFV p30 and p54 protein were used as targets and a phage-displayed 12-mer peptide library was used to conduct four rounds of biopanning to screen peptide epitopes, then amino acids GGG was used as a linker to synthesize tandem-epitope peptide of ASFV p30 and p54 protein which was used as coating antigen. The optimum reaction conditions of indirect ELISA were determined by chessboard titration, and clinical serum samples were used to evaluate the specificity, sensitivity, stability and conformity of this method. The biopanning experiment indicated that ¹⁴⁶PAEPYTT¹⁵² was a core domain of the B cell linear epitope of p54 protein. The optimization results of ELISA reaction conditions showed that the tandem-epitope peptide coupled with ovalbumin (OVA) at N-terminal had low background of non-specific serum reaction. And the optimum reaction effect was obtained when the polypeptide antigen was coated with carbonate buffer in 2 μg·mL^-1, the serum was diluted 100-fold with blocking solution (1% gelatin solution), and the HRP-antibody was diluted 5 000 times with 0.05% PBST solution. The cut-off value was determined to be 0.339. Furthermore, the results of specificity, sensitivity and stability tests showed that there is no cross-reaction in positive serum samples of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine pseudorabies virus (PRV), the detection limit of ASFV positive sera is 1∶1 600, and the method had high repeatability. Finally, Total 320 swine serum samples were detected simultaneously by the present established method and commercial ASFV antibody detection kit. The results showed that the relative specificity and sensitivity of the two methods were 97.6% and 97.3%, respectively. And the coincidence rate was 97.5%. In conclusion, this method showed good specificity, sensitivity, repeatability and coincidence rate, that had the potential value of developing clinical diagnostic kit.

Key words: African swine fever virus, p30 protein, p54 protein, biopinning, tandem-epitope peptide, indirect ELISA

中图分类号:

马俊, 王志远, 梁杏玲, 郑泽中, 杨汉春, 张桂红, 王衡. 基于非洲猪瘟病毒p30与p54蛋白表位串联多肽的间接ELISA抗体检测方法的建立[J]. 畜牧兽医学报, 2022, 53(12): 4325-4336.

MA Jun, WANG Zhiyuan, LIANG Xingling, ZHENG Zezhong, YANG Hanchun, ZHANG Guihong, WANG Heng. Development of an Indirect ELISA Antibodies Detection Method on Tandem-epitope Peptide of African Swine Fever Virus p30 and p54 Proteins[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(12): 4325-4336.

参考文献 26

[1]	BLOME S, GABRIEL C, BEER M. Pathogenesis of African swine fever in domestic pigs and European wild boar[J]. Virus Res, 2013, 173(1):122-130.
[2]	BURRAGE T G. African swine fever virus infection in Ornithodoros ticks[J]. Virus Res, 2013, 173(1):131-139.
[3]	SALGUERO F J. Comparative pathology and pathogenesis of African swine fever infection in swine[J]. Front Vet Sci, 2020, 7:282.
[4]	MONTGOMERY R E. On a form of swine fever occurring in British east Africa (Kenya Colony)[J]. J Comparat Pathol Therapeut, 1921, 34:159-191.
[5]	FAUQUET C M, MAYO M A, MANILOFF J, et al. Virus taxonomy:VIIIth report of the international committee on taxonomy of viruses[M]. San Diego:Academic Press, 2005:135-141.
[6]	PENRITH M L, KIVARIA F M, MASEMBE C. One hundred years of African swine fever:a tribute to R. Eustace Montgomery[J]. Transbound Emerg Dis, 2021, 68(5):2640-2642.
[7]	ZHOU X T, LI N, LUO Y Z, et al. Emergence of African swine fever in China, 2018[J]. Transbound Emerg Dis, 2018, 65(6):1482-1484.
[8]	BLOME S, FRANZKE K, BEER M. African swine fever-a review of current knowledge[J]. Virus Res, 2020, 287:198099.
[9]	NEILAN J G, ZSAK L, LU Z, et al. Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection[J]. Virology, 2004, 319(2):337-342.
[10]	LITHGOW P, TAKAMATSU H, WERLING D, et al. Correlation of cell surface marker expression with African swine fever virus infection[J]. Vet Microbiol, 2014, 168(2-4):413-419.
[11]	DIXON L K, SÁNCHEZ-CORDÓN P J, GALINDO I, et al. Investigations of pro- and anti-apoptotic factors affecting African swine fever virus replication and pathogenesis[J]. Viruses, 2017, 9(9):241.
[12]	SUN E C, ZHANG Z J, WANG Z L, et al. Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020[J]. Sci China Life Sci, 2021, 64(5):752-765.
[13]	SUN E C, HUANG L Y, ZHANG X F, et al. Genotype I African swine fever viruses emerged in domestic pigs in China and caused chronic infection[J]. Emerg Microbes Infect, 2021, 10(1):2183-2193.
[14]	PEÉREZ-FILGUEIRA D M, GONZAÁLEZ-CAMACHO F, GALLARDO C, et al. Optimization and validation of recombinant serological tests for African swine fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni Larvae[J]. J Clin Microbiol, 2006, 44(9):3114-3121.
[15]	GIMÉNEZ-LIROLA L G, MUR L, RIVERA B, et al. Detection of African swine fever virus antibodies in serum and oral fluid specimens using a recombinant protein 30 (p30) dual matrix indirect ELISA[J]. PLoS One, 2016, 11(9):e0161230.
[16]	GAO Z, SHAO J J, ZHANG G L, et al. Development of an indirect ELISA to specifically detect antibodies against African swine fever virus:bioinformatics approaches[J]. Virol J, 2021, 18(1):97.
[17]	曹琛福. 基于非洲猪瘟病毒P54蛋白抗原表位的ELISA检测方法建立及应用[D]. 广州:华南农业大学, 2016.CAO C F. Establishment and application of cELISA for detection of African swine fever based on p54 protein epitopes[D] Guangzhou: South China Agricultural University, 2016.(in Chinese)
[18]	陈孝军, 马俊, 陈熊男, 等. 1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定[J]. 畜牧兽医学报, 2022, 53(7): 2239-2251.CHEN X J, MA J, CHEN X N, et al. Identification of the B cell epitopes recognized by a monoclonal antibody against the p30 protein of African swine fever virus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2239-2251.(in Chinese)
[19]	SÁNCHEZ-VIZCAÍNO J M, MUR L, GOMEZ-VILLAMANDOS J C, et al. An update on the epidemiology and pathology of African swine fever[J]. J Comp Pathol, 2015, 152(1):9-21.
[20]	SÁNCHEZ-VIZCAÍNO J M, MUR L. African swine fever diagnosis update[J]. Dev Biol, 2013, 135:159-165.
[21]	ARIAS M, SÁNCHEZ-VIZCAÍNO J M. African swine fever eradication:the Spanish model[M]//MORILLA A, YOON K J, ZIMMERMAN J. Trends in Emerging Viral Infections of Swine. Ames, IA, USA:Iowa State Press, 2002:133-139.
[22]	GALLARDO C, FERNÁNDEZ-PINERO J, ARIAS M. African swine fever (ASF) diagnosis, an essential tool in the epidemiological investigation[J]. Virus Res, 2019, 271:197676.
[23]	GALLARDO C, REIS A L, KALEMA-ZIKUSOKA G, et al. Recombinant antigen targets for serodiagnosis of African swine fever[J]. Clin Vaccine Immunol, 2009, 16(7):1012-1020.
[24]	WU P, LOWE A D, RODRÍGUEZ Y Y, et al. Antigenic regions of African swine fever virus phosphoprotein p30[J]. Transbound Emerg Dis, 2020, 67(5):1942-1953.
[25]	MURGIA M V, MOGLER M, CERTOMA A, et al. Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus[J]. Arch Virol, 2019, 164(2):359-370.
[26]	高瞻. 非洲猪瘟病毒P30和P54蛋白抗原表位的分析及应用[D]. 北京:中国农业科学院, 2021.GAO Z. Analysis and application of African swine fever virus p30 and p54 protein antigenic epitopes [D]. Beijing: Chinese Academy of Agricultural Sciences, 2021(in Chinese)

基于非洲猪瘟病毒p30与p54蛋白表位串联多肽的间接ELISA抗体检测方法的建立

Development of an Indirect ELISA Antibodies Detection Method on Tandem-epitope Peptide of African Swine Fever Virus p30 and p54 Proteins

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 26

相关文章 15

编辑推荐

Metrics

本文评价

[1]	刘传霞, 王晓, 李雪雯, 鲍苗菲, 李婷婷, 陈欣, 翁长江, 郑君. 非洲猪瘟病毒pE120R蛋白单克隆抗体的制备[J]. 畜牧兽医学报, 2024, 55(1): 388-394.
[2]	王慧, 冯保亮, 吴丹, 向光明, 王楠, 牟玉莲, 李奎, 刘志国. CD163基因在猪繁殖与呼吸综合征抗病育种中的研究进展[J]. 畜牧兽医学报, 2023, 54(8): 3127-3138.
[3]	冯永智, 龚婷, 吴东东, 高琦, 郑晓宇, 张桂红, 孙彦阔. 影响非洲猪瘟病毒对培养细胞感染性的因素分析[J]. 畜牧兽医学报, 2023, 54(8): 3406-3414.
[4]	刘桃雪, 苏冰倩, 齐艳丽, 郭江涛, 刘忠虎, 褚贝贝, 王江, 曾磊. 非洲猪瘟病毒p30蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(8): 3415-3423.
[5]	丁晓艳, 何久香, 周晓杨, 周伃欣, 李晋涛. 非洲猪瘟病毒感染相关调控基因以及毒力基因初步筛选[J]. 畜牧兽医学报, 2023, 54(7): 2964-2971.
[6]	王映, 朱家宏, 赵加凯, 纪品品, 陈旭, 张路, 刘宝元, 孙亚妮, 赵钦. 抗非洲猪瘟病毒NP419L蛋白纳米抗体的筛选鉴定及其在抗体检测中的初步应用[J]. 畜牧兽医学报, 2023, 54(6): 2509-2520.
[7]	刘文豪, 朱彦策, 张冬萱, 王智豪, 张超. 稳定表达非洲猪瘟病毒E165R蛋白PK 15细胞系的构建[J]. 畜牧兽医学报, 2023, 54(6): 2662-2666.
[8]	王国超, 赵亚茹, 张忠辉, 张玉龙, 白鸽, 耿抒贤, 樊洁, 杨吉飞, 关贵全, 殷宏, 罗建勋, 牛庆丽. 非洲猪瘟病毒RNA聚合酶亚基D205R基因生物信息学分析及多克隆抗体制备[J]. 畜牧兽医学报, 2023, 54(5): 2042-2049.
[9]	陈杨, 孟林春, 郭梦娇, 张成成, 薄宗义, 楚电峰, 曹永忠, 吴艳涛, 张小荣. 检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2062-2072.
[10]	张婷, 冯涛, 杨金柯, 郝雨, 杨行, 张大俊, 史喜绢, 闫文倩, 陈玲玲, 刘湘涛, 郑海学, 张克山. 条件性敲除D1133L基因的重组非洲猪瘟病毒的构建及增殖特性[J]. 畜牧兽医学报, 2023, 54(2): 706-714.
[11]	张芳源, 杨大为, 仇德洋, 姜国骞, 李桂梅, 单虎. 非洲猪瘟病毒P30蛋白的表达及抗体液相芯片检测方法的建立[J]. 畜牧兽医学报, 2023, 54(10): 4300-4310.
[12]	齐艳丽, 刘桃雪, 于海深, 张超, 鲁维飞, 王江, 褚贝贝, 张改平. 非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2023, 54(1): 281-292.
[13]	王洋, 崔帅, 鑫婷, 王西西, 于海男, 陈世钰, 蒋亚君, 高新桃, 庞忠宝, 姜一曈, 郭晓宇, 贾红, 朱鸿飞. 非洲猪瘟病毒MGF360-14L靶向MAVS抑制Ⅰ型干扰素的产生[J]. 畜牧兽医学报, 2022, 53(9): 3272-3278.
[14]	赵旭阳, 靳家鑫, 路闻龙, 张帅, 黄丽, 张改平, 孙爱军, 庄国庆. 非洲猪瘟病毒免疫逃逸分子机制研究进展[J]. 畜牧兽医学报, 2022, 53(7): 2074-2082.
[15]	陈孝军, 马俊, 陈熊男, 梁祎凡, 高琦, 黄钊, 许润达, 郑佳琛, 郑泽中, 张桂红, 王衡, 龚浪. 1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定[J]. 畜牧兽医学报, 2022, 53(7): 2239-2251.