新型冠状病毒受体结合结构域分子探针的构建及在中和抗体分离中的应用

Construction of SARS-CoV-2 receptor binding domain molecular probe and its application in the isolation of neutralizing antibodies

目的构建新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)受体结合结构域(receptor binding domain,RBD)分子探针,对新型冠状病毒感染康复者(简称“新冠康复者”)的外周血单核细胞(peripheral blood mononuclear cells,PBMCs)进行单克隆记忆B细胞分选,获得RBD特异性中和抗体。方法从GenBank中下载SARS-CoV-2 RBD序列,在C末端加入Avi标签和6组氨酸标签后进行密码子优化设计、合成基因后克隆入表达载体pDRVI1.0,转染293F表达出蛋白进行生物素化后,利用该探针从2名新冠康复者的PBMCs中分选出单克隆RBD特异性B细胞。对B细胞裂解后,进行抗体轻重链可变区扩增,将获得的抗体轻重链基因分别克隆入表达载体,转染293F细胞后采用Protein A对抗体进行纯化,并利用SARS-CoV-2假病毒对其中和能力进行测试。结果成功获得了RBD-Avi蛋白,RBD-Avi探针生物素化效率达到30%~50%,Western Blot实验证实生物素化探针可以被来自于新冠康复者血浆中的抗体所识别,利用该探针可从2名新冠康复者体内成功分别分离出7个、16个RBD特异性记忆B细胞,分别占总细胞群的0.24%和0.17%;对获得的B细胞进行裂解后扩增出抗体的轻重链可变区,分别获得了7对、12对抗体轻重链。2名新冠康复者体内共表达出16个抗体,纯化抗体后利用SARS-CoV-2假病毒进行测试,大部分抗体对假病毒都展示出了中和活性,其中6个抗体的半数抑制浓度(half maximal inhibitory concentration,IC50)低于1μg/mL;XJ-A9和SCF-F1针对野生型假病毒的IC50值分别达到0.07μg/mL和0.35μg/mL。结论本研究构建的SARS-CoV-2 RBD分子探针具有良好的抗原性,分离的抗体具有针对SARS-CoV-2野生型假病毒的中和活性,具备深入研究的价值。

Objective To construct SARS-CoV-2 receptor binding domain molecular probe for monoclonal memory B cell sorting and obtain RBD specific neutralizing antibodies from peripheral blood mononuclear cells(PBMCs)of COVID-19 convalescents by single-cell sorting.Methods The SARS-CoV-2 RBD sequence was downloaded from GenBank,and the Avi tag and 6-histidine tags were added at the C-terminal.After codon optimization,it was chemically synthesized,cloned into the pDRVI1.0 vector,expressed after transfection of 293F cells,and biotinylated consequently.RBD-specific B cells were sorted out with this probe1 from the PBMCs of convalescents recovered from COVID-19.After B cells were lysed,the variable regions of heavy chain and light chain were amplified,cloned into the antibody expression vector,and transfected into 293F cells to express the antibody.Then the antibody was purified from the supernatant using protein A column and SARS-CoV-2 pseudovirus was used to test their neutralizing activity.Results RBD-Avi probe was produced and successfully biotinylated sequentially with an efficiency of 30%-50%.Western blot analysis revealed that the biotinylated probe was recognized by the antibodies purified from COVID-19 convalescent plasma.Using this probe,7 and 16 RBD-specific memory B cells were successfully isolated from the PBMCs of two convalescent individuals,accounting for 0.24%and 0.17%of the total cell population,respectively.After amplifying the variable regions of antibody heavy and light chains from the lysed B cells,7 and 12 pairs of antibody heavy-light chains were obtained.A total of 16 antibodies were expressed in the convalescent individuals,and most of the purified antibodies showed neutralizing activity against the pseudovirus,with IC50 values of 6 antibodies below 1µg/mL.The IC50 values of XJ-A9 and SCF-F1 against the wild-type pseudovirus were 0.07µg/mL and 0.35µg/mL,respectively.Conclusion The SARS-CoV-2 RBD molecular probe constructed in this study has good antigenicity,and the isolated antibodies present neutralizing activity against wild-type SARS-CoV-2 pseudovirus.