摘要
目的构建MR1/IgG1Fc融合蛋白杆状病毒表达载体并使其在昆虫细胞内表达获得MR1/IgG1Fc二聚体,制备人工抗原提呈细胞用于黏膜相关恒定T细胞(MAIT)反应性代谢物及MAIT细胞功能调控研究。方法从人外周血单个核细胞中扩增β2m编码基因。商业化合成MR1胞外段和IgG1Fc融合基因的全序列基因MR1/IgG1Fc,与β2m基因重组构建pFastBac^(TM)Dual+[β2m+MR1/IgG1Fc]表达载体;利用Bac-to-Bac昆虫表达系统表达MR1/IgG1Fc二聚体融合蛋白,并通过双抗体夹心ELISA、Western blot及流式细胞术进行检测。建立MR1/IgG1Fc二聚体加载的人工抗原提呈细胞(artificial antigen presenting cells,aAPCs),即MR1aAPCs,提呈大肠埃希菌(DH5α)来源的代谢物,与人外周血单个核细胞共培养,检测MAIT细胞的活化和增殖水平。结果经PCR及测序鉴定证实pFastBac^(TM)Dual+[β2m+MR1/IgG1Fc]载体中MR1/IgG1Fc与β2m具有正确序列。ELISA、Western blot及流式细胞术检测结果表明MR1/IgG1Fc二聚体在重组杆粒感染的Sf9细胞培养上清富集,具有正确构象。加载DH5α来源代谢物的MR1aAPCs能够促进MAIT细胞活化和增殖。结论成功构建pFastBac^(TM)Dual+[β2m+MR1/IgG1Fc]重组质粒,并在Sf9细胞中表达MR1/IgG1Fc二聚体,制备获得MR1aAPCs,为研究MAIT细胞反应性代谢物及MAIT细胞功能提供了新的工具。
Objective To construct the baculovirus expression vector of MR1/IgG1Fc fusion gene and express it in insect cells to obtain MR1/IgG1Fc dimer,and to prepare artificial antigen presenting cells to screen the reactivity metabolites of mucosal associated constant T(MAIT)cells and to study the regulatory function of MAIT cells.Methods Genes encoding human MR1and CH1-CH3regions of human IgG1andβ2mgene were amplified and merged as MR1/IgG1Fc.The recombinant vector pFastBac^(TM) Dual+[β2m+MR1/IgG1Fc]was constructed by inserting MR1/IgG1Fc andβ2minto two downstream promoters of Pph and Pp10in baculovirus expression vector pFastBac^(TM) Dual,respectively.Dimeric MR1/IgG1Fc fusion protein(MR1/IgG1Fc dimer)was expressed by Bac-to-Bac insect expression system,and detected by sandwich ELISA,Western blotting and flow cytometry.MR1/IgG1Fc dimer-loaded artificial antigen presenting cells(MR1aAPCs)were prepared.Metabolites derived fromE.coli DH5αwere extracted and pulsed onto MR1aAPCs and co-cultured with human peripheral blood mononuclear cells,followed by MAIT cells activation and proliferation detection.Results It was confirmed by PCR and sequencing that MR1/IgG1Fc andβ2mwere in correct sequence in pFastBac^(TM) Dual+[β2m+MR1/IgG1Fc]vector.The results of ELISA,Western blot and flow cytometry showed that MR1/IgG1Fc dimer was expressed in the supernatant of Sf9cells infected with recombinant bacmids,and the expressed fusion protein had correct conformation.MR1aAPCs loaded with DH5α-derived metabolites could promote MAIT cell activation and proliferation.Conclusion pFastBac^(TM) Dual+[β2m+MR1/IgG1Fc]recombinant plasmid is successfully constructed to express MR1/IgG1Fc dimer in Sf9cells.MR1aAPCs are prepared,which could be new research tools for studying MAIT cell reactive metabolites and MAIT cell function.
作者
赵桂华
龚业莉
翁秀芳
Zhao Guihua;Gong Yeli;Weng Xiufang(Department of Immunology,School of Medicine,Jianghan University,Wuhan 430056,China;Department of Immunology,School of Basic Medicine,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2023年第5期649-655,共7页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.32170920)
江汉大学校级自然科学基金项目(N0.2021yb135)。
