抗犬瘟热病毒犬源化嵌合单链抗体scFv-Fc的制备与活性分析

doi:10.11843/j.issn.0366-6964.2024.12.038

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (12): 5762-5773.doi: 10.11843/j.issn.0366-6964.2024.12.038

抗犬瘟热病毒犬源化嵌合单链抗体scFv-Fc的制备与活性分析

刘雅坤¹(), 毕振威²^,³^,*(), 夏兴霞²^,³, 莫菲²^,³, 徐司雨²^,³, 钱晶²^,³, 谭业平²^,³, 诸玉梅²^,³, 赵建军¹^,*()

1. 黑龙江八一农垦大学动物科技学院, 大庆 163319
2. 江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室, 南京 210014
3. 兽用生物制品(泰州)国泰技术创新中心, 泰州 225300

收稿日期:2024-02-04 出版日期:2024-12-23 发布日期:2024-12-27
通讯作者: 毕振威,赵建军 E-mail:910386496@qq.com;bizhenwei@126.com;zhaojianjun@byau.edu.cn
作者简介:刘雅坤(1998-), 女, 黑龙江鸡东人, 硕士生, 主要从事兽医病毒学与免疫学研究, E-mail: 910386496@qq.com
基金资助:
国家重点研发计划项目(2022YFD1800603)

Preparation and Activity Analysis of Canine Derived Chimeric Single Chain Antibody scFv-Fc against Canine Distemper Virus

LIU Yakun¹(), BI Zhenwei²^,³^,*(), XIA Xingxia²^,³, MO Fei²^,³, XU Siyu²^,³, QIAN Jing²^,³, TAN Yeping²^,³, ZHU Yumei²^,³, ZHAO Jianjun¹^,*()

1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China
2. Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
3. GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals, Taizhou 225300, China

Received:2024-02-04 Online:2024-12-23 Published:2024-12-27
Contact: BI Zhenwei, ZHAO Jianjun E-mail:910386496@qq.com;bizhenwei@126.com;zhaojianjun@byau.edu.cn

摘要/Abstract

摘要：

为减少和改善鼠源单克隆抗体在犬临床治疗中的异源性和功效，用RT-PCR方法从分泌抗犬瘟热病毒(CDV)中和单克隆抗体的鼠源杂交瘤细胞中扩增抗体重链可变区(V_H)和轻链可变区(V_L)，通过Linker将V_H和V_L连接获得单链抗体(scFv)基因，选择犬源抗体IgG B恒定区(Fc)连接到scFv的C末端获得犬源化嵌合单链抗体(scFv-Fc)基因；分别构建鼠源scFv与犬源化scFv-Fc的原核表达载体，通过大肠杆菌表达、纯化，用ELISA、IPMA、细胞融合抑制和中和试验检测表达抗体的特异性和中和活性。结果显示：CDV鼠源scFv、犬源化scFv-Fc的ELISA最低检出浓度分别为14.38和102.75 μg·mL^-1；与表达的CDV H蛋白和CDV均发生特异性反应；能够抑制CDV H和F蛋白介导的细胞膜融合，完全保护细胞不出现病变的最低浓度分别为0.72和0.64 μg·mL^-1。本研究成功制备了鼠源scFv与犬源化scFv-Fc嵌合抗体，为犬瘟热的临床治疗提供了候选抗体药物。

关键词: 犬瘟热病毒, 中和活性, 单链抗体, 嵌合抗体

Abstract:

To reduce and improve the heterogeneity and efficacy of mouse-derived monoclonal antibodies in clinical treatment of dogs, the variable region of heavy chain (V_H) and variable region of light chain (V_L) were amplified from mouse-derived hybridomas secreting monoclonal antibody against canine distemper virus (CDV) using RT-PCR, the single chain antibody (scFv) gene was obtained by linking V_H and V_L through Linker, and the canine-derived chimeric single chain antibody (scFv-Fc) gene was obtained by selecting the constant region (Fc) of canine IgG B to connect to the C-terminus of scFv; The prokaryotic expression vectors for expressing mouse-derived scFv and canine-derived scFv-Fc were respectively constructed, which were expressed in Escherichia coli for purification, and the specificity and neutralizing activity of the expressed antibodies were detected by ELISA, IPMA, cell fusion inhibition, and neutralization assay. The detection limit of ELISA for CDV mouse-derived scFv and canine-derived scFv-Fc are 14.38 and 102.75 μg·mL^-1, respectively; Both of scFv and scFv-Fc specifically react with the expressed CDV H protein and CDV; The cell membrane fusion mediated by CDV H and F proteins can be inhibited by scFv and scFv-Fc, and the minimum concentrations of them for complete protection of cells from CPE formation are 0.72 and 0.64 μg·mL^-1, respectively. The mouse-derived scFv and canine-derived chimeric antibody scFv-Fc are successfully developed, providing candidate antibody drugs for the clinical treatment of canine distemper.

Key words: canine distemper virus, neutralizing activity, single chain antibody, chimeric antibody

中图分类号:

S852.43

刘雅坤, 毕振威, 夏兴霞, 莫菲, 徐司雨, 钱晶, 谭业平, 诸玉梅, 赵建军. 抗犬瘟热病毒犬源化嵌合单链抗体scFv-Fc的制备与活性分析[J]. 畜牧兽医学报, 2024, 55(12): 5762-5773.

LIU Yakun, BI Zhenwei, XIA Xingxia, MO Fei, XU Siyu, QIAN Jing, TAN Yeping, ZHU Yumei, ZHAO Jianjun. Preparation and Activity Analysis of Canine Derived Chimeric Single Chain Antibody scFv-Fc against Canine Distemper Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5762-5773.

图/表 7

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参考文献 34

1	BEINEKE A , BAUMGÄRTNER W , WOHLSEIN P . Cross-species transmission of canine distemper virus-an update[J]. One Health, 2015, 1, 49- 59. doi: 10.1016/j.onehlt.2015.09.002
2	DEEM S L , SPELMAN L H , YATES R A , et al. Canine distemper in terrestrial carnivores: a review[J]. J Zoo Wildl Med, 2000, 31 (4): 441- 451. doi: 10.1638/1042-7260(2000)031[0441:CDITCA]2.0.CO;2
3	COSBY S L . Morbillivirus cross-species infection: is there a risk for humans?[J]. Fut Virol, 2012, 7 (11): 1103- 1113. doi: 10.2217/fvl.12.103
4	RENDON-MARIN S , DA FONTOURA BUDASZEWSKI R , CANAL C W , et al. Tropism and molecular pathogenesis of canine distemper virus[J]. Virol J, 2019, 16 (1): 30. doi: 10.1186/s12985-019-1136-6
5	BEINEKE A , PUFF C , SEEHUSEN F , et al. Pathogenesis and immunopathology of systemic and nervous canine distemper[J]. Vet Immunol Immunopathol, 2009, 127 (1-2): 1- 18. doi: 10.1016/j.vetimm.2008.09.023
6	LEMPP C , SPITZBARTH I , PUFF C , et al. New aspects of the pathogenesis of canine distemper leukoencephalitis[J]. Viruses, 2014, 6 (7): 2571- 2601. doi: 10.3390/v6072571
7	MARTELLA V , BLIXENKRONE-MøLLER M , ELIA G , et al. Lights and shades on an historical vaccine canine distemper virus, the Rockborn strain[J]. Vaccine, 2011, 29 (6): 1222- 1227. doi: 10.1016/j.vaccine.2010.12.001
8	ZHANG H , SHAN F , ZHOU X , et al. Outbreak and genotyping of canine distemper virus in captive Siberian tigers and red pandas[J]. Sci Rep, 2017, 7 (1): 8132. doi: 10.1038/s41598-017-08462-4
9	MARTELLA V , ELIA G , BUONAVOGLIA C . Canine distemper virus[J]. Vet Clin North Am Small Anim Pract, 2008, 38, 787- 797. doi: 10.1016/j.cvsm.2008.02.007
10	ANIS E , HOLFORD A L , GALYON G D , et al. Antigenic analysis of genetic variants of Canine distemper virus[J]. Vet Microbiol, 2018, 219, 154- 160. doi: 10.1016/j.vetmic.2018.03.014
11	KRUMM S A , YAN D , HOVINGH E S , et al. An orally available, small-molecule polymerase inhibitor shows efficacy against a lethal morbillivirus infection in a large animal model[J]. Sci Transl Med, 2014, 6 (232): 232ra52.
12	TREJO-AVILA L M , MORALES-MARTÍNEZ M E , RICQUE-MARIE D , et al. In vitro anti-canine distemper virus activity of Fucoidan extracted from the brown alga Cladosiphon okamuranus[J]. VirusDisease, 2014, 25 (4): 474- 480. doi: 10.1007/s13337-014-0228-6
13	潘阳, 王露楠. 单克隆抗体人源化技术研究进展[J]. 国际检验医学杂志, 2011, 32 (13): 1483- 1484.
	PAN Y , WANG L N . Research progress on humanization technology of monoclonal antibodies[J]. International Journal of Laboratory Medicine, 2011, 32 (13): 1483- 1484.
14	赵丹, 邱冬, 韩德敏, 等. H₃N₂亚型犬流感病毒鼠/犬嵌合抗体scFv-Fc的制备与活性分析[J]. 南京农业大学学报, 2019, 42 (3): 474- 481.
	ZHAO D , QIU D , HAN D M , et al. Preparation and activity of murine/canine chimeric antibody scFv-Fc against H₃ N₂ canine influenza virus[J]. Journal of Nanjing Agricultural University, 2019, 42 (3): 474- 481.
15	周丽璇. 抗犬细小病毒嵌合抗体c10H4的构建及治疗性评价[D]. 郑州: 河南农业大学, 2022.
	ZHOU L X. Construction and therapeutic evaluation of chimeric antibody c10H4 against canine parvovirus[D]. Zhengzhou: Henan Agricultural University, 2022. (in Chinese)
16	WANG W J , BI Z W , LIU Y K , et al. Development of a monoclonal antibody recognizing novel linear neutralizing epitope on H protein of canine distemper virus vaccine strains (America-1 genotype)[J]. Int J Biol Macromol, 2023, 246, 125584. doi: 10.1016/j.ijbiomac.2023.125584
17	毕振威. 犬瘟热病毒单克隆抗体的制备及其应用[D]. 南京: 南京农业大学, 2012.
	BI Z W. Preparation and application of the monoclonal antibodies against canine distemper virus[D]. Nanjing: Nanjing Agricultural University, 2012. (in Chinese)
18	孙婧. 犬瘟热病毒单克隆抗体夹心ELISA检测方法和胶体金免疫层析试纸条的研究[D]. 泰安: 山东农业大学, 2013.
	SUN J. Study of the monoclonal antibodies-based sandwich ELISA and colloidal gold immunochromatographic strip for the detection of canine distemper virus[D]. Tai'an: Shandong Agricultural University, 2013. (in Chinese)
19	罗雯, 阎岩, 徐志凯, 等. 鼠源单抗3G1单链抗体基因的构建及序列分析[J]. 西安联合大学学报, 2003, 6 (2): 5- 8.
	LUO W , YAN Y , XU Z K , et al. Construction and sequence analysis of the single chain Fv of mouse monoclonal antibody 3G1[J]. Journal of Xi'an United University, 2003, 6 (2): 5- 8.
20	AVILA M , ALVES L , KHOSRAVI M , et al. Molecular determinants defining the triggering range of prefusion F complexes of canine distemper virus[J]. J Virol, 2014, 88 (5): 2951- 2966. doi: 10.1128/JVI.03123-13
21	BI Z W , WANG W J , XIA X X . Structure and function of a novel lineage-specific neutralizing epitope on H protein of canine distemper virus[J]. Front Microbiol, 2022, 13, 1088243.
22	王志明. 转基因小鼠技术在全人源抗体药物研发中的应用[J]. 中国新药杂志, 2016, 25 (22): 2596- 2602.
	WANG Z M . Application of transgenic mouse technology platform in the discovery and development of full human antibody drugs[J]. Chinese Journal of New Drugs, 2016, 25 (22): 2596- 2602.
23	杨郑欣, 李琰, 张晓茜, 等. 单个B细胞抗体制备技术研究进展[J]. 中国兽医杂志, 2023, 59 (8): 82- 87.
	YANG Z X , LI Y , ZHANG X Q , et al. Research progress on single B cell antibody preparation technology[J]. Chinese Journal of Veterinary Medicine, 2023, 59 (8): 82- 87.
24	LUO D , MAH N , KRANTZ M , et al. Vl-linker-Vh orientation-dependent expression of single chain Fv containing an engineered disulfide-stabilized bond in the framework regions[J]. J Biochem, 1995, 118 (4): 825- 831. doi: 10.1093/oxfordjournals.jbchem.a124986
25	HUSTON J S , LEVINSON D , MUDGETT-HUNTER M , et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli[J]. Proc Natl Acad Sci USA, 1988, 85 (16): 5879- 5883. doi: 10.1073/pnas.85.16.5879
26	WANG Y , YUAN W J , GUO S Q , et al. A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments[J]. Nat Commun, 2022, 13 (1): 4614. doi: 10.1038/s41467-022-32423-9
27	ZHANG T , WANG C Y , ZHANG W , et al. Generation and characterization of a fusion protein of single-chain fragment variable antibody against hemagglutinin antigen of avian influenza virus and truncated protamine[J]. Vaccine, 2010, 28 (23): 3949- 3955. doi: 10.1016/j.vaccine.2010.03.045
28	JUN H R , PHAM C D , LIM S I , et al. An RNA-hydrolyzing recombinant antibody exhibits an antiviral activity against classical swine fever virus[J]. Biochem Biophys Res Commun, 2010, 395 (4): 484- 489. doi: 10.1016/j.bbrc.2010.04.032
29	ZIMMERMANN J , SAALBACH I , JAHN D , et al. Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens[J]. BMC Biotechnol, 2009, 9, 79.
30	PYO H M , KIM I J , KIM S H , et al. Escherichia coli expressing single-chain Fv on the cell surface as a potential prophylactic of porcine epidemic diarrhea virus[J]. Vaccine, 2009, 27 (14): 2030- 2036. doi: 10.1016/j.vaccine.2009.01.130
31	赵肃清, 林明霞, 劳志婷, 等. 一种G11-scFv-Nluc双功能活性的融合蛋白及其应用: 中国, 113563484B[P]. 2023-04-21.
	ZHAO S Q, LIN M X, SU L T, et al. A fusion protein with dual functional activity of G11 scFv Nluc and its application: CN, 113563484B[P]. 2023-04-21. (in Chinese)
32	PYZIK M , SAND K M K , HUBBARD J J , et al. The neonatal Fc receptor (FcRn): a misnomer?[J]. Front Immunol, 2019, 10, 1540. doi: 10.3389/fimmu.2019.01540
33	CHALLA D K, VELMURUGAN R, OBER R J, et al. FcRn: from molecular interactions to regulation of IgG pharmacokinetics and functions[M]//DAERON M, NIMMERJAHN F. Fc Receptors. Cham: Springer, 2014: 249-272.
34	BERGERON L M , MCCANDLESS E E , DUNHAM S , et al. Comparative functional characterization of canine IgG subclasses[J]. Vet Immunol Immunopathol, 2014, 157 (1-2): 31- 41. doi: 10.1016/j.vetimm.2013.10.018

引物对名称 Names of prime pairs	引物对序列(5′→3′) Sequences of prime pairs
mouse-V_H-F	CCGAGGCGCAGCTGCAGGAGTCTGG
mouse-V_H-R	GTGGTCCCTTGGCCCC
mouse-V_L-F	GACATTGTGATGACGCAGTCTCC
mouse-V_L-R	ACGTTTGATCTCCACCTTGGTCCC
G12-V_H-F	CCG$\underline{{\rm{GAATTC}}}$GAGGTTACTCTAAAAG(横线示EcoR Ⅰ)
G12-V_H-R	CC$\underline{{\rm{AAGCTT}}}$TGAGGAGACGGTG(横线示Hind Ⅲ)
G12-Linker-V_L-F	$\underline{{\rm{AAGCTT}}}$GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGAT- ATTGT(横线示Hind Ⅲ，斜体为Linker序列)
G12-V_L-R	CCG$\underline{{\rm{CTCGAG}}}$TTTTTATTTCCAGCTT(横线示Xho I)
dogC_H-F	$\underline{{\rm{ACCAAGCTGGAAATAAAA}}}$accgtctcctcagcctcc(横线示同源臂)
dogC_H-R	$\underline{{\rm{CAGTGGTGGTGGTGGTGGTGCTCGAG}}}$tcatttacccggagaatg(横线示同源臂)

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抗犬瘟热病毒犬源化嵌合单链抗体scFv-Fc的制备与活性分析

Preparation and Activity Analysis of Canine Derived Chimeric Single Chain Antibody scFv-Fc against Canine Distemper Virus

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