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

doi:10.11843/j.issn.0366-6964.2023.06.029

摘要/Abstract

摘要： 非洲猪瘟(African swine fever, ASF)是猪的一种高度传染性疾病,严重威胁全球养猪业的发展。本研究旨在筛选抗非洲猪瘟病毒(ASF virus, ASFV)NP419L蛋白的纳米抗体并对其在ASF抗体检测中的应用进行初步评价。大肠杆菌表达和Ni-NTA亲和层析纯化等技术表达纯化重组NP419L蛋白;将纯化的重组蛋白免疫双峰驼,5次免疫采集外周血淋巴细胞提取总RNA,经反转录、巢式PCR、酶切、连接和电转化等构建抗NP419L蛋白的噬菌体展示文库;利用噬菌体展示筛淘技术,经三轮筛淘获得抗NP419L蛋白的纳米抗体;构建纳米抗体与辣根过氧化物酶(HRP)融合蛋白的真核表达载体,转染HEK293T细胞,ELISA和间接免疫荧光(IFA)等鉴定融合蛋白的分泌表达及其与NP419L蛋白的结合;竞争ELISA初步评价获得的纳米抗体与HRP融合蛋白在ASF抗体检测中的应用。结果显示,成功制备了纯度较高的预期大小为48 ku的重组NP419L蛋白;构建获得了阳性率为89.5%,库容量为6×10⁸的噬菌体展示文库;筛淘获得了6株抗NP419L蛋白的纳米抗体;分泌表达了该6株纳米抗体与HRP的融合蛋白,ELISA检测发现该6株融合蛋白可特异性结合重组NP419L蛋白,IFA检测发现1株融合蛋白与真核表达的NP419L蛋白结合,该融合蛋白可作为探针应用于ASF抗体的检测。本研究成功筛选出6株抗ASFV NP419L蛋白的特异性纳米抗体,并对其进行了结合蛋白特性的鉴定和抗体检测初步应用的评价,为后续ASF诊断技术的研发和NP419L蛋白功能研究奠定了基础。

关键词: 非洲猪瘟病毒, NP419L蛋白, 纳米抗体

Abstract: African swine fever (ASF) is a highly infectious disease of pigs, which seriously threatens the development of the global pig industry. The purpose of this study is to screen specific nanobodies against the NP419L protein of ASF virus (ASFV) and evaluate its application of anti-ASFV antibody detection. We prepared the recombinant NP419L protein by E.coli expression system and Ni-NTA affinity chromatography purification. The purified recombinant protein was used to immunize the Bactrian camels. After the fifth immunization, peripheral blood lymphocytes were collected and total RNA was extracted. A phage display library against NP419L protein was constructed by nested PCR, enzyme digestion, ligation and electroporation. Nanobodies against NP419L were obtained through three rounds of screening using phage display screening technology. The eukaryotic expression vectors of nanobodies fused with horseradish peroxidase (HRP) proteins were constructed and transfected into HEK293T cells. The secretory expression of the fusion protein and its binding to NP419L protein were identified by ELISA and indirect immunofluorescence (IFA), and preliminary evaluation of the obtained nanobody fused with HRP proteins in ASF detection using competitive ELISA. The results showed that the NP419L recombinant protein with the expected size of 48 ku was successfully prepared with high purity; a phage display library with a positive rate of 89.5% and a library capacity of 6×10⁸was constructed; six strains of anti-NP419L protein nanobodies were obtained by screening; the fusion proteins of these six nanobodies with HRP were secretively expressed, the results of ELISA showed that the six fusion proteins specifically bind to the recombinant NP419L protein, one fusion protein was found to bind to NP419L protein expressed with eukaryotic system by IFA, and this fusion protein can be used as a probe for the detection of ASFV antibodies. In the study, six nanobodies against ASFV NP419L protein were successfully screened. The preliminary characterization of its binding protein and its application of antibody detection were also carried out, which provided the basis for the subsequent development of ASF diagnostic technology and the functional study of NP419L protein.

Key words: African swine fever virus, NP419L protein, nanobody

中图分类号:

S852.659.1

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.

WANG Ying, ZHU Jiahong, ZHAO Jiakai, JI Pinpin, CHEN Xu, ZHANG Lu, LIU Baoyuan, SUN Yani, ZHAO Qin. Screening and Identification of Nanobodies against NP419L Protein of African Swine Fever Virus and Its Preliminary Application of Antibody Detection[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2509-2520.

参考文献 34

[1]	DIXON L K, SUN H, ROBERTS H. African swine fever[J]. Antiviral Res, 2019, 165:34-41.
[2]	WANG F X, ZHANG H, HOU L N, et al. Advance of African swine fever virus in recent years[J]. Res Vet Sci, 2021, 136:535-539.
[3]	GE S Q, LI J M, FAN X X, et al. Molecular characterization of African swine fever virus, China, 2018[J]. Emerg Infect Dis, 2018, 24(11):2131-2133.
[4]	URBANO A C, FERREIRA F. African swine fever control and prevention:an update on vaccine development[J]. Emerg Microbes Infect, 2022, 11(1):2021-2033.
[5]	DIXON L K, CHAPMAN D A G, NETHERTON C L, et al. African swine fever virus replication and genomics[J]. Virus Res, 2013, 173(1):3-14.
[6]	WANG G G, XIE M J, WU W, et al. Structures and functional diversities of ASFV proteins[J]. Viruses, 2021, 13(11):2124.
[7]	AKAIKE T. Role of free radicals in viral pathogenesis and mutation[J]. Rev Med Virol, 2001, 11(2):87-101.
[8]	FORMAN H J, TORRES M. Redox signaling in macrophages[J]. Mol Aspects Med, 2001, 22(4-5):189-216.
[9]	REDREJO-RODRÍGUEZ M, GARCÍA-ESCUDERO R, YÁÑEZ-MUÑOZ R J, et al. African swine fever virus protein pE296R is a DNA repair apurinic/apyrimidinic endonuclease required for virus growth in swine macrophages[J]. J Virol, 2006, 80(10):4847-4857.
[10]	REDREJO-RODRÍGUEZ M, ISHCHENKO A A, SAPARBAEV M K, et al. African swine fever virus AP endonuclease is a redox-sensitive enzyme that repairs alkylating and oxidative damage to DNA[J]. Virology, 2009, 390(1):102-109.
[11]	OLIVEROS M, YÁÑEZ R J, SALAS M L, et al. Characterization of an African swine fever virus 20-kDa DNA polymerase involved in DNA repair[J]. J Biol Chem, 1997, 272(49):30899-30910.
[12]	YÁÑEZ R J, VIÑUELA E. African swine fever virus encodes a DNA ligase[J]. Virology, 1993, 193(1):531-536.
[13]	CHEN Y Q, LIU H H, YANG C, et al. Structure of the error-prone DNA ligase of African swine fever virus identifies critical active site residues[J]. Nat Commun, 2019, 10(1):387.
[14]	WAN Y, SHI Z W, PENG G, et al. Development and application of a colloidal-gold dual immunochromatography strip for detecting African swine fever virus antibodies[J]. Appl Microbiol Biotechnol, 2022, 106(2):799-810.
[15]	PETROVAN V, MURGIA M V, WU P, et al. Epitope mapping of African swine fever virus (ASFV) structural protein, p54[J]. Virus Res, 2020, 279:197871.
[16]	HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMANS S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature, 1993, 363(6428):446-448.
[17]	MUYLDERMANS S. Nanobodies:natural single-domain antibodies[J]. Annu Rev Biochem, 2013, 82:775-797.
[18]	MUYLDERMANS S. Applications of nanobodies[J]. Annu Rev Anim Biosci, 2021, 9:401-421.
[19]	XU J L, XU K, JUNG S, et al. Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants[J]. Nature, 2021, 595(7866):278-282.
[20]	HE L, TAI W B, LI J F, et al. Enhanced ability of oligomeric nanobodies targeting MERS coronavirus receptor-binding domain[J]. Viruses, 2019, 11(2):166.
[21]	HARMSEN M M, FIJTEN H P D, DEKKER A, et al. Passive immunization of pigs with bispecific llama single-domain antibody fragments against foot-and-mouth disease and porcine immunoglobulin[J]. Vet Microbiol, 2008, 132(1-2):56-64.
[22]	CARDOSO F M, IBAÑEZ L I, VAN DEN HOECKE S, et al. Single-domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge[J]. J Virol, 2014, 88(15):8278-8296.
[23]	ZHU M, GONG X, HU Y H, et al. Streptavidin-biotin-based directional double Nanobody sandwich ELISA for clinical rapid and sensitive detection of influenza H5N1[J]. J Transl Med, 2014, 12:352.
[24]	ZHAO J K, ZHU J H, WANG Y, et al. A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus[J]. Virol Sin, 2022, 37(6):922-933.
[25]	ZHAO H J, REN J H, WU S Y, et al. HRP-conjugated-nanobody-based cELISA for rapid and sensitive clinical detection of ASFV antibodies[J]. Appl Microbiol Biotechnol, 2022, 106(11):4269-4285.
[26]	SHENG Y M, WANG K, LU Q Z, et al. Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay[J]. J Nanobiotechnol, 2019, 17(1):35.
[27]	刘青源. 基于纳米抗体-HRP融合蛋白鸡血清中抗NDV抗体竞争ELISA检测方法的建立[D]. 杨凌:西北农林科技大学, 2019.LIU Q Y. Establishment of a competitive ELISA assay for anti-NDV antibody in chicken serum based on nanobody-HRP fusion protein[D]. Yangling: Northwest A&F University, 2019. (in Chinese)
[28]	刘红亮. 猪繁殖与呼吸综合征病毒Nsp9蛋白纳米抗体的筛选及其抗病毒功能研究[D]. 杨凌:西北农林科技大学, 2016.LIU H L. Isolation and antiviral activities analysis of porcine peproductive and respiratory syndrome virus Nsp9- specific nanobodies[D]. Yangling: Northwest A&F University, 2016. (in Chinese)
[29]	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.
[30]	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.
[31]	DUAN H, CHEN X, ZHAO J K, et al. Development of a nanobody-based competitive enzyme-linked immunosorbent assay for efficiently and specifically detecting antibodies against genotype 2 porcine reproductive and respiratory syndrome viruses[J]. J Clin Microbiol, 2021, 59(12):e0158021.
[32]	MU Y, JIA C Y, ZHENG X, et al. A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2[J]. J Nanobiotechnol, 2021, 19(1):34.
[33]	LU Q Z, LI X X, ZHAO J K, et al. Nanobody-horseradish peroxidase and-EGFP fusions as reagents to detect porcine parvovirus in the immunoassays[J]. J Nanobiotechnol, 2020, 18(1):7.
[34]	JI P P, ZHU J H, LI X X, et al. Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus[J]. J Nanobiotechnol, 2020, 18(1):44.