猪繁殖与呼吸综合征病毒GP5蛋白纳米抗体的筛选及其对病毒复制的抑制效应

doi:10.11843/j.issn.0366-6964.2024.01.024

摘要/Abstract

摘要： 为获得猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）GP5蛋白的特异性纳米抗体，并探究其对病毒复制的抑制效应。使用原核表达系统将PRRSV-GP5蛋白大量表达后经镍柱亲和层析法纯化，使用所获的重组GP5蛋白免疫羊驼，并于第0、14、28、42 天时采血，检测羊驼体内抗体效价后分离全血淋巴细胞，提取细胞总RNA，反转录后使用巢式PCR扩增出重链抗体可变区（VHH）片段，并与pCANTAB-5E载体相连后转化入TG1感受态细胞，利用噬菌体展示技术构建GP5-VHH噬菌体抗体展示文库。经连续三轮特异性噬菌体的筛选与富集后，淘选出与PRRSV-GP5蛋白高结合力的纳米抗体，通过间接ELISA方法鉴定其反应原性，后将所获的纳米抗体基因克隆至真核表达载体pcDNA3.1（-）中，利用Lipofectamine3000转染至Marc-145细胞内，与PRRSV分别共孵育0、24、36、48、60、72 h，以探究所筛针对PRRSV-GP5蛋白的纳米抗体于胞内对病毒复制与转录产生的影响。结果表明，成功对PRRSV-GP5蛋白进行原核表达并纯化，得到浓度为2.16 mg·mL^-1的重组蛋白，对羊驼三次免疫14 d后，其体内抗体效价高达1∶819 200，构建GP5-VHH噬菌体抗体展示文库，库容量达2.93×10⁷ CFU·mL^-1，插入率为98%。经对噬菌体抗体文库连续三轮淘选富集后，最终获得2株不同氨基酸序列的纳米抗体。间接ELISA结果显示，2株纳米抗体均与PRRSV-GP5蛋白具有良好的亲和力。将2株纳米抗体转染至Marc-145细胞内，它们分别于36与60 h时显著阻碍PRRSV的复制与转录，展现出了较好的阻碍病毒复制的能力。本研究首次筛选出针对PRRSV-GP5蛋白的特异性纳米抗体，且经验证所筛纳米抗体具备抑制PRRSV在细胞内复制的能力，研究结果为抗PRRSV新型药物的研发奠定了试验依据与物质基础。

关键词: 猪繁殖与呼吸综合征病毒, GP5蛋白, 纳米抗体, 噬菌体展示技术, 病毒复制

Abstract: In order to obtain specific nanobodies against GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV) and explore their inhibitory effect on virus replication. The PRRSV-GP5 protein was expressed in a large amount by prokaryotic expression system and purified by nickel column affinity chromatography. The obtained recombinant GP5 protein was used to immunize alpacas, and blood was collected at the 0th, 14th, 28th, and 42nd days. After detecting the antibody titer in alpacas, the whole blood lymphocytes were isolated, and the total cell RNA was extracted. After reverse transcription, the heavy chain antibody variable region (VHH) fragment was amplified by nested PCR, and connected with pCANTAB-5E vector, and then transformed into TG1 receptor cells, The GP5-VHH phage antibody display library was constructed using phage display technology. After three rounds of screening and enrichment of specific phages, nanobodies with high affinity to PRRSV-GP5 protein were screened out, and their reactivity was identified by indirect ELISA. To investigate the effects of screened nanobodies targeting PRRSV-GP5 protein on virus replication and transcription in the cell,the obtained nanobody genes were cloned into eukaryotic expression vector pcDNA3.1 (-) and transfected into Marc-145 cells using Lipofectamine3000. They were incubated with PRRSV for 0, 24, 36, 48, 60, and 72 hours, respectively. The results showed that the PRRSV-GP5 protein was successfully expressed and purified in prokaryotic form, and the recombinant protein with a concentration of 2.16 mg·mL^-1 was obtained. After 14 days of three immunizations, the antibody titer in alpaca was as high as 1∶819 200. The GP5-VHH phage antibody display library was constructed, with a capacity of 2.93×10⁷ CFU·mL^-1, the insertion rate was 98%. After three consecutive rounds of panning and enrichment of phage antibody library, two nanobodies with different amino acid sequences were finally obtained. The results of indirect ELISA showed that the two nanobodies had good affinity with PRRSV-GP5 protein. Two nanobodies were transfected into Marc-145 cells, and they significantly hindered the replication and transcription of PRRSV at 36 and 60 hours,respectively.They demonstrated good ability to inhibit virus replication. In this study, the specific nanobodies against PRRSV-GP5 protein was screened for the first time and it has been verified that the screened nanobodies have the ability to inhibit PRRSV replication in cells. The research results could lay the experimental and material foundation for the development of new drugs against PRRSV.

Key words: porcine reproductive and respiratory syndrome virus, GP5 protein, nanobody, phage display technology, virus replication

中图分类号:

S852.659.6

宋雯妍, 张瀚文, 吴澳迪, 张丽燕, 刘照, 叶桐桐, 陈创夫, 盛金良. 猪繁殖与呼吸综合征病毒GP5蛋白纳米抗体的筛选及其对病毒复制的抑制效应[J]. 畜牧兽医学报, 2024, 55(1): 258-270.

SONG Wenyan, ZHANG Hanwen, WU Aodi, ZHANG Liyan, LIU Zhao, YE Tongtong, CHEN Chuangfu, SHENG Jinliang. Screening of Nanobodies against Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein and Exploration of Their Inhibitory Effect on Virus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 258-270.

参考文献 25

[1]	ALBINA E. Epidemiology of porcine reproductive and respiratory syndrome (PRRS):an overview[J]. Vet Microbiol, 1997, 55(1-4):309-316.
[2]	JOHNSON C R, GRIGGS T F, GNANANDARAJAH J, et al. Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative ORF5 present in all arteriviruses[J]. J Gen Virol, 2011, 92(Pt 5):1107-1116.
[3]	BALKA G, PODGÓRSKA K, BRAR M S, et al. Genetic diversity of PRRSV 1 in central eastern Europe in 1994-2014:origin and evolution of the virus in the region[J]. Sci Rep, 2018, 8(1):7811.
[4]	PEJSAK Z, MARKOWSKA-DANIEL I. Losses due to porcine peproductive and respiratory syndrome in a large swine farm[J]. Comp Immunol Microbiol Infect Dis, 1997, 20(4):345-352.
[5]	ZIEBUHR J, SNIJDER E J, GORBALENYA A E. Virus-encoded proteinases and proteolytic processing in the Nidovirales[J]. J Gen Virol, 2000, 81(Pt 4):853-879.
[6]	SNIJDER E J, KIKKERT M, FANG Y. Arterivirus molecular biology and pathogenesis[J]. J Gen Virol, 2013, 94(Pt 10):2141-2163.
[7]	THAA B, SINHADRI B C, TIELESCH C, et al. Signal peptide cleavage from GP5 of PRRSV:a minor fraction of molecules retains the decoy epitope, a presumed molecular cause for viral persistence[J]. PLoS One, 2013, 8(6):e65548.
[8]	CHUEH L L, LEE K H, WANG F I, et al. Sequence analysis of the nucleocapsid protein gene of the porcine reproductive and respiratory syndrome virus Taiwan MD-001 strain[M]//ENJUANES L, SIDDELL S G, SPAAN W. Coronaviruses and Arteriviruses. Boston:Springer, 1998:795-799.
[9]	VERHEIJE M H, WELTING T J M, JANSEN H T, et al. Chimeric arteriviruses generated by swapping of the M protein ectodomain rule out a role of this domain in viral targeting[J]. Virology, 2002, 303(2):364-373.
[10]	DELPUTTE P L, VANDERHEIJDEN N, NAUWYNCK H J, et al. Involvement of the matrix protein in attachment of Porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages[J]. J Virol, 2002, 76(9):4312-4320.
[11]	HAMERS-CASTERMAN C, ATARHOUCH T, MUYLDERMANS S, et al. Naturally occurring antibodies devoid of light chains[J]. Nature, 1993, 363(6428):446-448.
[12]	CHAKRAVARTY R, GOEL S, CAI W B. Nanobody:the "magic bullet" for molecular imaging?[J]. Theranostics, 2014, 4(4):386-398.
[13]	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)
[14]	张勋, 赵庆亮, 杨素芳, 等. 1株NSP2蛋白5个氨基酸缺失的PRRSV遗传进化分析[J]. 西北农林科技大学学报:自然科学版, 2017, 45(2):37-43.ZHANG X, ZHAO Q L, YANG S F, et al. Genetic variation analysis of a porcine reproductive and respiratory syndrome virus strain with 5 amino acids deletion in NSP2 protein[J]. Journal of Northwest A&F University:Natural Science Edition, 2017, 45(2):37-43. (in Chinese)
[15]	刘建奎, 徐叶, 刘辰, 等. 基于全基因组分析2017-2021年福建省猪繁殖与呼吸综合征病毒基因组特征[J]. 畜牧兽医学报, 2023, 54(4):1579-1589.LIU J K, XU Y, LIU C, et al. Genomic characteristics of porcine reproductive and respiratory syndrome virus in Fujian province from 2017 to 2021 based on whole genome[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4):1579-1589. (in Chinese)
[16]	刘晓东, 杨旭兵, 王福军, 等. PRRSV流行毒株遗传变异分析[J]. 家畜生态学报, 2017, 38(7):66-70.LIU X D, YANG X B, WANG F J, et al. Genetic variation analysis on the PRRSV strain isolated[J]. Acta Ecologiae Animalis Domastici, 2017, 38(7):66-70. (in Chinese)
[17]	WANG Y, GUO J, QIAO S, et al. GP5 protein-based ELISA for the detection of PRRSV antibodies[J]. Pol J Vet Sci, 2016, 19(3):495-501.
[18]	DU L P, YU Z Y, PANG F J, et al. Targeted delivery of GP5 antigen of PRRSV to M cells enhances the antigen-specific systemic and mucosal immune responses[J]. Front Cell Infect Microbiol, 2018, 8:7.
[19]	JOBLING S A, JARMAN C, TEH M M, et al. Immunomodulation of enzyme function in plants by single-domain antibody fragments[J]. Nat Biotechnol, 2003, 21(1):77-80.
[20]	MEI Y X, CHEN Y Z, SIVACCUMAR J P, et al. Research progress and applications of nanobody in human infectious diseases[J]. Front Pharmacol, 2022, 13:963978.
[21]	鲍奕恺. 靶向PSMA多价纳米抗体的制备及其偶联药物的毒性评价[D]. 无锡:江南大学, 2022.BAO Y K. Production of multivalent anti-PSMA nanobodies and the toxicity evaluation of nanobody-drug conjugate[D]. Wuxi:Jiangnan University, 2022. (in Chinese)
[22]	KIM J Y J, SANG Z, XIANG Y F, et al. Nanobodies:robust miniprotein binders in biomedicine[J]. Adv Drug Deliv Rev, 2023, 195:114726.
[23]	宋欢, 孙明霞, 尹坤, 等. 猪繁殖与呼吸综合征病毒Nsp2蛋白纳米抗体的筛选及其鉴定[J]. 中国预防兽医学报, 2019, 41(4):385-390.SONG H, SUN M X, YIN K, et al. Identification and screening of porcine reproductive and respiratory syndrome virus Nsp2-specific nanobodies[J]. Chinese Journal of Preventive Veterinary Medicine, 2019, 41(4):385-390. (in Chinese)
[24]	DELHALLE S, SCHMIT J C, CHEVIGNÉ A. Phages and HIV-1:From display to interplay[J]. Int J Mol Sci, 2012, 13(4):4727-4794.
[25]	REPERANT L A, OSTERHAUS A D M E. AIDS, Avian flu, SARS, MERS, Ebola, Zika... what next?[J]. Vaccine, 2017, 35(35):4470-4474.