高表达H2型人类组织血型抗原HEK-293T细胞系的构建及其应用  

Construction and application of HEK-293T cells with high expression of H2 histo-blood group antigen

作　　者：秦海艳 谢忆 张巧玲 马素娟 李晨 杨林鹏 付艳丽 李奇蒙 杨俊杰 QIN Haiyan;XIE Yi;ZHANG Qiaoling;MA Sujuan;LI Chen;YANG Linpeng;FU Yanli;LI Qimeng;YANG Junjie(Lanzhou Institute of Biological Products Co.,Ltd.,Gansu Provincial Vaccine Technology Innovation Center,Lanzhou 730046,Gansu Province,China)

机构地区：[1]兰州生物制品研究所有限责任公司、甘肃省疫苗技术创新中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2024年第4期9-15,共7页Progress In Microbiology and Immunology

基　　金：甘肃省科技重大专项(17ZD2FA007)。

摘　　要：目的通过在人胚肾-293T(human embryo kidney-293T,HEK-293T)细胞中过表达岩藻糖转移酶2(fucosyltransferases 2,FUT2)基因,进而催化诺如病毒受体人类组织血型抗原(histo-blood group antigen,HBGA)的形成,构建介导诺如病毒和受体发生相互作用的细胞系。方法构建FUT2慢病毒质粒,采用Lipofectamine 2000将FUT2慢病毒质粒及pMD.2G和psPAX2包装质粒共转染HEK-293T细胞,收获慢病毒颗粒。慢病毒颗粒感染对数生长期的HEK-293T细胞,嘌呤霉素加压筛选获得HEK-293T/FUT2细胞系。PCR鉴定FUT2基因是否整合到基因组中,RT-qPCR检测HEK-293T/FUT2细胞FUT2 mRNA转录水平,流式细胞术分析HEK-293T/FUT2细胞HBGAs的表达,间接免疫荧光法分析HEK-293T/FUT2细胞与诺如病毒病毒样颗粒(virus-like particle,VLP)的结合活性。结果成功构建了序列正确的慢病毒质粒PLVX-IRES-Puro-FUT2,并获得了慢病毒颗粒。慢病毒颗粒感染HEK-293T细胞后,通过嘌呤霉素加压筛选获得了HEK-293T/FUT2细胞系,PCR验证显示,FUT2基因成功整合到HEK-293T细胞基因组中。HEK-293T/FUT2细胞FUT2 mRNA水平提高了330倍。99.9%的HEK-293T/FUT2细胞表达H2型人类组织血型抗原。GI.1重组诺如病毒VLP可以与HEK-293T/FUT2细胞产生很好的结合反应,EC50为2.007μg/mL。结论建立了H2型人类组织血型抗原稳定高表达的HEK-293T细胞系,并基于该细胞系初步建立了GI.1型重组诺如病毒VLP结合活性评价方法。Objective To construct a new cell line by overexpressing fucosyltransferases 2(FUT2)gene in human embryo kidney-293T(HEK-293T)cells,FUT2 has the founction of catalyzing the formation of Histo-blood group antigen(HBGA),which is one of receptors of norovirus.The constructed cell line was used to study interactions of norovirus and its receptor.Methods FUT2 lentivirus plasmid was constructed,and the FUT2 lentivirus plasmid,pMD.2G and psPAX2 packaging plasmids were co-transfected into HEK-293T cells with Lipofectamine 2000.Lentivirus particles were harvested and infected HEK-293T cells at logarithmic growth stage,to obtain HEK-293T/FUT2 cell lines by purinomycin pressure screening.PCR was used to identify whether the FUT2 gene was integrated into the genome.The mRNA transcription level of FUT2 in HEK-293T/FUT2 cells was detected by RT-qPCR,and the expression of HBGAs in HEK-293T/FUT2 cells was analyzed by flow cytometry.Further more,the binding activity of characterized HEK-293T/FUT2 cells with norovirus virus-like particles(VLP)was analyzed by indirect immunofluorescence.Results The lentivirus plasmid PLVX-IRES-Puro-FUT2 with correct sequence was successfully constructed and lentivirus particles were obtained.HEK-293T/FUT2 cell lines were obtained after lentivirus particles were infected into HEK-293T cells.PCR verification showed that FUT2 gene was successfully integrated into the genome of HEK-293T cells.The level of FUT2 mRNA in HEK-293T/FUT2 cells was increased by 330 times,and 99.9%of HEK-293T/FUT2 cells expressed H2 Histo-blood group antigen.Finally,GI.1 type recombinant norovirus VLP were able to bind to HEK-293T/FUT2 cells,and EC_(50)was 2.007μg/mL.Conclusion In this study,HEK-293T/FUT2 cell line with stable and high expression of H2 Histo-blood group antigen was constructed for the first time,and a preliminary evaluation method for the detection of GI.1 recombinant norovirus VLP binding activity was established based on this cell line.

关 键 词：人类组织血型抗原 人胚肾293T细胞(HEK-293T细胞) 诺如病毒 α-1 2岩藻糖转移酶 结合活性 

分 类 号：R373.3[医药卫生—病原生物学]