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

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^(-1)45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∶819200,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^(-1)45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。

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^(-1)45 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∶819200.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^(-1)45 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.