畜牧兽医学报2024，Vol.55Issue(1)：258-270,13.DOI:10.11843/j.issn.0366-6964.2024.01.024
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
Screening of Nanobodies against Porcine Reproductive and Respiratory Syndrome Virus GP5 Protein and Exploration of Their Inhibitory Effect on Virus Replication
摘要
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 puri-fied 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 detec-ting 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 nanobod-ies 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 in-to 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 successful-ly 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 al-paca 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 nano-bodies 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.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Key words
porcine reproductive and respiratory syndrome virus/GP5 protein/nanobody/phage display technology/virus replication分类
农业科技引用本文复制引用
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):258-270,13.基金项目
国家自然科学基金(31960691) （31960691）
自治区研究生科研创新项目(XJ2022G119) （XJ2022G119）
