猫杯状病毒VP1蛋白的单克隆抗体制备及抗原表位鉴定

doi:10.11843/j.issn.0366-6964.2024.12.040

摘要/Abstract

摘要：

本研究旨在制备并鉴定针对猫杯状病毒(feline calicivirus，FCV) VP1蛋白的单克隆抗体(monoclonal antibodies, mAbs)。本研究使用SH14株FCV全病毒作为免疫原，通过腹腔注射法免疫BALB/c小鼠，制备了针对VP1蛋白的mAb。通过ELISA筛选，成功获得特异性单克隆抗体3A3C1株。对3A3C1株mAb进行了全面的生物学特性鉴定，3A3C1株mAb确认为IgG₁型，轻链为κ链。Western blot和间接免疫荧光检测的结果显示，3A3C1株mAb能特异性地识别并结合FCV SH14株、F9株和GZ22。此外，本研究还深入探究了3A3C1株mAb的线性抗原表位。通过分段表达VP1蛋白并进行详细的Western blot分析，最终确定3A3C1株mAb的线性抗原表位位于VP1蛋白的⁴²⁶PSRLTPAGDYAITSG⁴⁴⁰区域。本研究制备的3A3C1株mAb不仅是理解FCV免疫学特性的重要工具，也对发展FCV诊断方法和疫苗策略具有潜在应用价值。

关键词: 猫杯状病毒, 单克隆抗体, 抗原表位, 衣壳蛋白

Abstract:

The purpose of this study was to prepare and identify monoclonal antibodies (mAbs) against the VP1 protein of feline calicivirus (FCV) strain SH14. In this study, the whole virus of the SH14 strain of FCV was used as an immunogen to immunize BALB/c mice through intraperitoneal injection, and mAbs against the VP1 protein were prepared. Through ELISA screening, a specific monoclonal antibody clone, 3A3C1, was successfully obtained. A comprehensive biological characterization of the 3A3C1 clone mAb was conducted, confirming it as an IgG₁ type with a κ light chain. The results of Western blot and immunofluorescence assay (IFA) showed that the 3A3C1 clone mAb could specifically recognize and bind to FCV strains SH14, F9, and GZ22. In addition, this study further explored the linear antigenic epitope of the 3A3C1 clone mAb. By segmentally expressing the VP1 protein and conducting detailed Western blot analysis, the linear antigenic epitope of the 3A3C1 clone mAb was finally determined to be located in the⁴²⁶PSRLTPAGDYAITSG⁴⁴⁰ region of the VP1 protein. The 3A3C1 clone mAb prepared in this study is not only an important tool for understanding the immunological characteristics of FCV but also has potential applications in developing FCV diagnostic methods and vaccine strategies.

Key words: feline calicivirus, monoclonal antibody, antigenic epitope, capsid protein

中图分类号:

S852.659.6

张泽宇, 董宁宁, 谈晓梅, 李传锋, 朱杰, 刘光清, 张伟, 孟春春. 猫杯状病毒VP1蛋白的单克隆抗体制备及抗原表位鉴定[J]. 畜牧兽医学报, 2024, 55(12): 5784-5791.

ZHANG Zeyu, DONG Ningning, TAN Xiaomei, LI Chuanfeng, ZHU Jie, LIU Guangqing, ZHANG Wei, MENG Chunchun. Preparation of Monoclonal Antibodies against Feline Calicivirus VP1 Protein and Identification of Antigenic Epitopes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5784-5791.

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参考文献 20

1	CASTRO T X , CUBEL GARCIA RDE C , FUMIAN T M , et al. Detection and molecular characterization of caliciviruses (vesivirus and norovirus) in an outbreak of acute diarrhea in kittens from Brazil[J]. Vet J, 2015, 206 (1): 115- 117. doi: 10.1016/j.tvjl.2015.06.014
2	HOFMANN-LEHMANN R , HOSIE M J , HARTMANN K , et al. Calicivirus infection in cats[J]. Viruses, 2022, 14 (5): 937. doi: 10.3390/v14050937
3	RADFORD A D , COYNE K P , DAWSON S , et al. Feline calicivirus[J]. Vet Res, 2007, 38 (2): 319- 335. doi: 10.1051/vetres:2006056
4	KAISER W J , CHAUDHRY Y , SOSNOVTSEV SV , et al. Analysis of protein-protein interactions in the feline calicivirus replication complex[J]. J Gen Virol, 2006, 87 (Pt 2): 363- 368.
5	赵艳丽, 董宏伟, 陈小庆, 等. 猫杯状病毒基因及其抗原多样性研究进展[J]. 中国预防兽医学报, 2014, 36 (3): 251- 254. doi: 10.3969/j.issn.1008-0589.2014.03.20
	ZHAO Y L , DONG H W , CHEN X Q , et al. Progress in the study of feline calicivirus genes and their antigenic diversity[J]. Chinese Journal of Preventive Veterinary Medicine, 2014, 36 (03): 251- 254. doi: 10.3969/j.issn.1008-0589.2014.03.20
6	FUJITA S , KOBA R , TOHYA Y . Identification of amino acid substitutions escaping from a broadly neutralizing monoclonal antibody of feline calicivirus[J]. Vet Res, 2022, 318, 198848.
7	程晨曦, 刘家森, 胡晓亮, 等. 猫杯状病毒VP1蛋白单克隆抗体的制备及其抗原表位的鉴定[J]. 中国预防兽医学报, 2017, 29 (12): 1017- 1021.
	CHENG C X , LIU J S , HU X L , et al. Preparation of monoclonal antibodies against feline calicivirus VP1 protein and identification of its antigenic epitopes[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39 (12): 1017- 1021.
8	孙雅鑫, 吴斌. 猫杯状病毒及疾病诊疗研究进展[J]. 动物医学进展, 2015, 36 (11): 97- 101. doi: 10.3969/j.issn.1007-5038.2015.11.022
	SUN Y X , WU B . Research progress on feline calicivirus and its disease diagnosis and treatment[J]. Progress in Veterinary Medicine, 2015, 36 (11): 97- 101. doi: 10.3969/j.issn.1007-5038.2015.11.022
9	SEAL B S , RIDPATH J F , MENGELING W L . Analysis of feline calicivirus capsid protein genes: identification of variable antigenic determinant regions of the protein[J]. J Gen Virol, 1993, 74 (Pt 11): 2519- 2524.
10	OSSIBOFF R J , ZHOU Y , LIGHTFOOT P J , et al. Conformational changes in the capsid of a calicivirus upon interaction with its functional receptor[J]. J Virol, 2010, 84 (11): 5550- 5564. doi: 10.1128/JVI.02371-09
11	方水琴, 刘程, 马俊飞, 等. 小鼠杂交瘤单克隆抗体快速制备技术研究进展[J]. 生物工程学报, 2021, 37 (07): 2293- 2306.
	FANG S Q , LIU C , MA J F , et al. Research progress in rapid preparation techniques for mouse hybridoma monoclonal antibodies[J]. Chinese Journal of Biotechnology, 2021, 37 (07): 2293- 2306.
12	张路捷, 高雁怩, 夏婷婷, 等. 非洲猪瘟病毒p30蛋白单克隆抗体的制备与鉴定[J]. 畜牧与兽医, 2021, 53 (6): 111- 115.
	ZHANG L J , GAO Y N , XIA T T , et al. Preparation and identification of monoclonal antibodies against African swine fever virus p30 protein[J]. Animal Husbandry and Veterinary Medicine, 2021, 53 (06): 111- 115.
13	王欣荣, 张文立, 马文杰, 等. 识别不同亚型鸭甲型肝炎病毒VP0蛋白单克隆抗体的制备及表位鉴定[J]. 中国预防兽医学报, 2023, 45 (4): 416- 422.
	WANG X R , ZHANG W L , MA W J , et al. Preparation and epitope identification of monoclonal antibodies recognizing different subtypes of duck hepatitis A virus VP0 protein[J]. Chinese Journal of Preventive Veterinary Medicine, 2023, 45 (4): 416- 422.
14	TAN M , HEGDE R S , JIANG X . The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors[J]. J Virol, 2004, 78 (12): 6233- 6242. doi: 10.1128/JVI.78.12.6233-6242.2004
15	JUNG J , GRANT T , THOMAS D R , et al. High-resolution cryo-EM structures of outbreak strain human norovirus shells reveal size variations[J]. Proc Natl Acad Sci U S A, 2019, 116 (26): 12828- 12832. doi: 10.1073/pnas.1903562116
16	FANG H , TAN M , XIA M , et al. Norovirus P particle efficiently elicits innate, humoral and cellular immunity[J]. PLoS One, 2013, 8 (4): e63269. doi: 10.1371/journal.pone.0063269
17	CUBILLOS-ZAPATA C , ANGULO I , ALMANZA H , et al. Precise location of linear epitopes on the capsid surface of feline calicivirus recognized by neutralizing and non-neutralizing monoclonal antibodies[J]. Vet Res, 2020, 51 (1): 59. doi: 10.1186/s13567-020-00785-x
18	WANG X , XU F , LIU J , et al. Atomic model of rabbit hemorrhagic disease virus by cryo-electron microscopy and crystallography[J]. Plos Pathog, 2013, 9 (1): e1003132. doi: 10.1371/journal.ppat.1003132
19	GEISSLER K , SCHNEIDER K , TRUYEN U . Mapping neutralizing and non-neutralizing epitopes on the capsid protein of feline calicivirus[J]. J Vet Med B Infect Dis Vet Public Health, 2002, 49 (1): 55- 60. doi: 10.1046/j.1439-0450.2002.00529.x
20	TOHYA Y , TANIGUCHI Y , TSUBAKIMOTO M , et al. Preparation and characterization of neutralizing monoclonal antibodies to feline calicivirus[J]. Nihon Juigaku Zasshi, 1990, 52 (2): 251- 256. doi: 10.1292/jvms1939.52.251

引物名称 Primers	序列(5′→3′) Sequences	产物大小/bp Product size
P1(426-547)	F: ctcggtaccctcgagggatccCCGAGCCGTCTGACCCCG R: agactgcaggtcgacaagcttACGAACCACTTTGTCACGATCC	366
P2(547-668)	F: ctcggtaccctcgagggatccATTAGCGTGCTGCCGGAAA R: agactgcaggtcgacaagcttAGCCCGGGATCCCAGTTT	366
P3(386-611)	F: ctcggtaccctcgagggatccACCACCGCGACCATCAGC R: agactgcaggtcgacaagcttGATACGATAAACCGCCAGGCTGTCCGGCGCCAGCAGGT	378
P4(426-456)	F: ctcggtaccctcgagggatccCCGAGCCGTCTGACCCCG R: agactgcaggtcgacaagcttGATAAAGCCGGTGTTGCTAACTT	93
P5(456-486)	F: ctcggtaccctcgagggatccACCACCGCGACCATCAGC R: agactgcaggtcgacaagcttACGAACCACTTTGTCACGATCC	93
P6(441-471)	F: ctcggtaccctcgagggatccATTAAGAACAACACCAACTTCAAAAGC R: agactgcaggtcgacaagcttTTTGCTCACACGGTTGTCTTGG	93

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