新型冠状病毒S1、S2和NTD抗原特异性B细胞及其亚型标记技术的建立与应用  

Establishment and Application of A Labeling Method for S1⁃,S2⁃,and NTD Antigen⁃specific B Cells and Their Subtypes of SARS⁃CoV⁃2

作　　者：宋彦丽 杨惠洁 赵玉秀 鲍春婷 李淑艳 李长贵[2] SONG Yanli;YANG Huijie;ZHAO Yuxiu;BAO Chunting;LI Shuyan;LI Changgui(Research Division 2 of Viral Vaccines,Wuhan Institution of Biological Product Co.Ltd,Wuhan 430207,China;Research Division of Respiratory Virus Vaccine,National Institute for Food and Drug Control,Beijing 102600,China;The First Research Group,Beijing Institution of Biological Product Co.Ltd,Beijing 102600,China;Research Division of Viral Vaccines,Changchun Institution of Biological Product Co.Ltd,Changchun 130012,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究二室,武汉430207 [2]中国食品药品检定研究院呼吸道病毒疫苗室,北京102600 [3]北京生物制品研究所课题一组,北京100176 [4]长春生物制品研究所有限责任公司疫苗研究室,长春130012

出　　处：《病毒学报》2024年第4期679-686,共8页Chinese Journal of Virology

摘　　要：建立一种新型冠状病毒(Severe acute respiratory syndrome coronavirus 2,SARS⁃CoV⁃2)S1、S2、NTD蛋白特异性B细胞及其亚型的标记技术,该技术可通过与流式细胞术联用检测抗原特异性B细胞及其亚型含量,或与10×免疫组库联用对抗原特异性B细胞进行测序。首先,测试最佳蛋白使用浓度:将生物素标记的S1、S2和NTD蛋白按照600 nmol/L、1200 nmol/L、2400 nmol/L、3600 nmol/L和4800 nmol/L分别与荧光素⁃亲和素偶联,通过流式检测S1+CD19+B、S2+CD19+B、NTD+CD19+B细胞占比,确定不同蛋白对应的最佳使用浓度;进一步优化生物素⁃蛋白与荧光素⁃亲和素的比例:将检测生物素⁃蛋白与荧光⁃亲和素的摩尔比分别按照4∶1,5∶1和6∶1比例测试,对小鼠脾脏S1+CD19+B、S2+CD19+B、NTD+CD19+B细胞及S1+CD19+CD27+B、S2+CD19+CD27+B、NTD+CD19+CD27+B细胞的检测效果,优化荧光素⁃亲和素的最佳使用量;通过测试BV421、Super Bright 600、PE、PE/Cy7和APC等荧光对小鼠脾细胞的非特异结合能力,选择非特异结合较弱的荧光;根据筛选的荧光,给生物素化的S1、S2和NTD均标记PE和APC荧光,建立检测这3个蛋白特异性B细胞和记忆B细胞的方法,并测试带核酸序列的荧光素⁃亲和素对B细胞和记忆B细胞的检测效果,测试该方法的稳定性。S1、S2和NTD蛋白浓度分别为4800 nmol/L、3600 nmol/L和3600 nmol/L时,对B细胞的检测效果最佳,且对阴性小鼠的染色效果较低;生物素⁃S1蛋白与荧光素⁃亲和素的最佳摩尔比为6∶1时,检测结果最佳;生物素⁃S2蛋白、生物素⁃NTD蛋白与荧光素⁃亲和素均在4:1时,检测结果最佳,且在该浓度下BV421、APC、Super Bright 600、PE与细胞的非特异结合较弱;建立了一种检测SARS⁃CoV⁃2 S1、S2和NTD抗原特异性B细胞及记忆B细胞的方法,通过对一批样本的重复3次检测,发现该方法较为稳定,CV值≤20%。通过抗原特异性B细胞标记技术,实现了对SARS⁃CoV⁃2 S1�To establish a labeling method for spike(S)1⁃,S2⁃,and N⁃terminal domain(NTD)⁃specific B cells(and their subtypes)of severe acute respiratory syndrome coronavirus 2(SARS⁃CoV⁃2).This method could be used with flow cytometry to test,or in 10×immune repertoire sequence to obtain the sequence of antigen⁃specific B cells and their subtypes.First,S1,S2,and NTD proteins were coupled with fluorescein–streptavidin(600,1200,2400,3600 and 4800 nmol/L),respectively.Antigen⁃specific B cells were detected by flow cytometry to determine the optimal concentration.Second,the molar ratio of biotin–protein and fluorescein–streptavidin was tested at ratios of 4:1,5:1,and 6:1,respectively.According to the ratio of S1+cluster of differentiation(CD)19+B cells,S2+CD19+B cells,NTD+CD19+B cells,as well as S1+CD19+CD27+B cells,S2+CD19+CD27+B cells,and NTD+CD19+CD27+B cells,the optimal concentration was obtained.Third,the non⁃specific effects of different types of fluorescein(BV421,Super Bright 600,PE,PE/Cy7 and APC)were tested to evaluate their non⁃specific binding.Fourth,the established method was repeated thrice to test its stability in flow cytometry.We also tested it with fluorescein–avidin–biotin⁃containing nucleotide sequences which could be used in a 10×immune repertoire sequence.First,the optimal concentrations(in nmol/L)of S1,S2,and NTD proteins were 4800,3600,and 3600 nmol/L,respectively.Second,the optimal molar ratio of biotin–S1 protein to fluorescein–avidin was 6:1,whereas the most suitable molar ratio of biotin–S2 protein and biotin–NTD protein to fluorescein–avidin was 4:1,respectively.At this concentration,BV421,APC,Super Bright 600,and PE showed weak non⁃specific binding.Third,repeating this method thrice showed it to be highly stable,with a coefficient of variation of≤20%,and it was suitable for fluorescein–avidin–biotin⁃containing nucleotide sequences.The established labeling technology for S1⁃,S2⁃,and NTD protein⁃specific B cells(and their subtypes)of SARS⁃CoV

关 键 词：流式细胞术 生物素⁃蛋白 荧光素⁃亲和素 新型冠状病毒 抗原特异性B细胞 

分 类 号：R392-3[医药卫生—免疫学]