抗SARS-CoV-2刺突糖蛋白受体结合域单链抗体的筛选及鉴定  

Screening and identification of single-chain fragment variable against the receptor-binding domain of spike protein of SARS-CoV-2

作　　者：金佳佩 蔡雪飞[1] JIN Jiapei;CAI Xuefei(Key Laboratory of Molecular Biology on Infectious Diseases,Ministry of Education,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2023年第3期295-305,共11页Chinese Journal of Biologicals

基　　金：重庆市技术创新与应用发展专项重点项目(cstc2020jscx-dxwtBX0022);重庆医科大学新型冠状病毒(2019-nCoV)感染的肺炎应急研究专项(CQMUNCP0208)。

摘　　要：目的 构建抗严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-Co V-2)刺突糖蛋白(spike protein,S)受体结合域(receptor-binding domain,RBD)的单链抗体(single-chain fragment variable,scFv)噬菌体展示文库,筛选特异性scFv,并进行功能鉴定。方法 提取RBD蛋白免疫小鼠的脾细胞m RNA,逆转录为c DNA,以其为模板扩增scFv的重链可变区(hight chain fragment of variable,VH)和轻链可变区(light chain fragment of variable,VL)基因,经过重叠延伸PCR扩增(splicing overlap extension PCR,SOE-PCR)组装为scFv基因片段。将scFv基因片段插入噬菌体载体,构建scFv噬菌体展示文库。经4轮生物淘洗,筛选出与RBD结合能力较强的scFv基因,进行重组表达、纯化和生物活性鉴定。结果 构建的scFv噬菌体文库滴度为6.0×10^(11)pfu/m L。经4轮生物淘选后,共筛选出4株与RBD结合较强的scFv,分别为scFv11、scFv12、scFv25、scFv28。scFv相对分子质量约为27 000,多以包涵体形式存在,纯化后可与HRP标记的鼠抗His单克隆抗体发生特异性结合,浓度达2.4 mg/m L,纯度约90%。4株scFv均可与RBD重组蛋白发生特异性结合;除scFv28外,其他3株scFv均可与野生型和多突变株S蛋白结合;与RBD均存在剂量依赖性相互作用,亲和力动态拟合解离常数(K_(D))分别为8.9、5.92、10.67、2.36 nmol/L,稳态拟合解离常数(K_(D))分别为5.3、6.5、8.7、5.8 nmol/L;scFv11、scFv12和scFv25可同时识别3个独立的RBD多肽,包括RBD2(S^(334~353))、RBD9(S^(439~458))和RBD13(S^(499~518));在线服务器SWISS-MODEL对scFv进行同源建模的模型质量良好,可用于分子对接;scFv11及人肺泡上皮细胞表面血管紧张素转换酶2(human angiotensin converting enzyme 2,ACE2)与RBD相互作用的界面仅部分重合,scFv12和scFv25与RBD的相互作用界面和ACE2与RBD相互作用界面存在较大差异。结论 本研究通过构建抗SARS-Co V-2 RBD的scFv噬菌体文库,筛选并制备了可与SARS-Co V-2Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10^(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_(D)s)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_(D)s)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S^(334~353)),RBD9(S^(439~458))and RBD13(S^(499~518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at w

关 键 词：严重急性呼吸综合征冠状病毒2 单链抗体 刺突糖蛋白 受体结合域 噬菌体文库 

分 类 号：R373.1[医药卫生—病原生物学]