重组抗SARS-CoV-2 IgA单克隆抗体的优化表达及其初步鉴定

Expression optimization and preliminary identification of recombinant monoclonal antibody IgA against SARS-CoV-2

目的制备针对严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)IgA单克隆抗体,对其表达条件进行优化以提高表达量,并初步探讨IgA抗体在抗SARS-CoV-2中的效应。方法将编码IgA1-F61抗体序列的质粒与聚乙烯亚胺(polyethyleneimine,PEI)转染试剂混合后转染EXPi293F^(TM)细胞,瞬时表达抗体蛋白;通过优化重链(heavy chain,Hc)、轻链(light chain,Lc)、J链(joining chain,Jc)比例、质粒与PEI比例、转染后收获时间确定单体IgA1(monomeric IgA1,mIgA1)-F61及二聚体IgA1(dimeric IgA1,dIgA1)-F61在EXPi293F^(TM)细胞中的最佳培养条件以及最佳表达量。将转染后上清经亲和层析纯化后,BCA法测定浓度,SDS-PAGE及体积排阻高效液相色谱(size exclusion chromatography-high performance liquid chromatography,SEC-HPLC)法分析抗体表达完整性及纯度,假病毒中和试验检测抗体中和活性。结果当He与Lc比例为1:2、质粒与PEI比例为1:3、转染后5d收获上清时,mIgA1-F61最高表达量为123.45μg/mL,纯化后抗体纯度达95%以上;Hc:Lc:Jc为1:2:1时,可获得最大纯度的dIgA1-F61,达90%以上。针对OmicronBA.4/5的假病毒中和试验结果显示,dIgA1-F61呈现优于IgG-F61的中和活性,其最大半数抑制浓度(half maximal inhibitory concentration,IC_(50))降低了4倍左右。结论成功表达了重组抗SARS-CoV-2单克隆抗体mIgA1-F61和dIgA1-F61,纯度较高,且dIgA1在体外展现出优于IgG的中和活性。

Objective To prepare monoclonal antibody IgA against severe acute respiratory syndrome coronavirus 2(SARSCoV-2),optimize relevant expression conditions to increase the expression level,and preliminarily explore the effect of IgA antibody on anti-SARS-CoV-2.Methods The plasmid encoding IgAl-F61 antibody sequence was mixed with polyethyleneimine(PEI)transfection reagent and then transfected into EXPi293F~(TM)cells to transiently express antibody protein;The optimal culture conditions and expression levels of monomeric IgAl(mIgAl)-F61 and dimeric IgAl(dIgAl)-F61 in EXPi293F~(TM)cells were determined by optimizing the ratio of heavy chain(Hc),light chain(Lc)and joining chain(Jc),the proportion of plasmid and PEI,and the harvest time after transfection.The supernatant after transfection was purified by affinity chromatography,and then determined for the concentration by BCA,analyzed for the expression integrity and purity of antibody by SDS-PAGE and size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),and detected for the neutralizing activity of antibody by pseudovirus neutralization assay.Results The optimal expression level of mIgAl-F61 was 123.45μg/mL and the purity of purified antibody was over 95%when the ratio of Hc to Lc was 1:2,the ratio of plasmid to PEI was 1:3,and the supernatant was harvested 5 d after transfection;The highest purity of dIgAlF61 was more than 90%when the ratio of Hc:Lc:Jc was 1:2:1.The results of pseudovirus neutrali-zation assay against Omicron BA.4/5 showed that dIgAl-F61 exhibited better neutralizing activity than IgG-F61,and the value of half maximum inhibitory concentration(IC_(50))was reduced by about 4 times.Conclusion Recombinant monoclonal antibodies mIgAl-F61 and dIgAl-F61 against SARS-CoV-2 were successfully expressed with high purity and dIgAl showed better neutralizing activity than IgG in vitro.