β2微球蛋白-接头蛋白2-人类白细胞抗原A24(β2m-linker 2-HLA A24)蛋白的生物信息学分析、真核表达与鉴定  

Bioinformation analysis, eukaryotic expression and identification of β2m-linker 2-HLA A24

作　　者：高雯琬 陈思吟 徐敏 陶崑[1] GAO Wenwan;CHEN Siyin;XU Min;TAO Kun(Department of Immunology,College of Basic Medical Science,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学基础医学院免疫教研室,重庆400016

出　　处：《细胞与分子免疫学杂志》2020年第5期398-403,共6页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的应用生物信息学方法对β2微球蛋白-接头蛋白2-人类白细胞抗原A24(β2m-linker 2-HLA A24)蛋白进行结构、功能预测分析,并对该蛋白进行真核表达与纯化。方法运用在线蛋白质分析软件ProtParam预测β2m-linker 2-HLA A24蛋白理化性质;磷酸化位点预测软件NetPhos和蛋白质二级结构预测软件SOPMA分析其磷酸化位点和二级结构;并利用蛋白质结构模型预测工具SWISS-MODEL建立三级结构模型。构建含组氨酸标签(His tag)的重组质粒pcDNA3.4/β2m-linker 2-HLA A24-His tag,在Expi293F细胞表达,经镍柱亲和层析获得β2m-linker 2-HLA A24蛋白;采用SDS-PAGE、Western blot、间接ELISA验证蛋白的纯度和性质。结果β2m-linker 2-HLA A24蛋白含有44个磷酸化位点,属于亲水性蛋白,二级结构以无规卷曲为主,并成功构建三级结构模型;成功构建pcDNA3.4/β2m-linker 2-HLA A24-His tag重组质粒,并在Expi293F细胞表达系统中获得可溶性表达。结论成功构建β2m-linker 2-HLA A24蛋白,生物信息学结果显示该蛋白具备与抗原肽结合并激活T细胞的功能。为后续联合使用该蛋白与抗原肽激活抗原特异性CD8^+T细胞实验奠定了基础。Objective To explore the structure and function ofβ2 microglobulin-linker 2-human leukocyte antigen A24(β2m-linker 2-HLA A24)by bioinformatics,and to obtain the purified protein by eukaryotic expression.MethodsThe physicochemical properties ofβ2m-linker 2-HLA A24 was predicted by ProtParam.The predicting software of phosphorylation site(NetPhos)and the predicting software of the secondary structure(SOPMA)were used to analyze the phosphorylation site and the secondary structure of the protein,respectively.And the predicting tool of modeling the proteins’structure(SWISS-MODEL)was used to model its tertiary structure.Then the recombinant plasmid of pcDNA3.4/β2m-linker 2-HLA A24-His tag was constructed and transfected into Expi293F cells.The protein of interest was purified through Ni-affinity chromatography.And the purity and properties of the protein were identified by SDS-PAGE,Western blot analysis and indirect ELISA.Results Theβ2m-linker 2-HLA A24 protein contained 44 phosphorylated sites.The protein was predicted to be a hydrophilic protein,of which the secondary structure mainly consists of irregular curl,and the modeling of the tertiary structure was successful.The recombinant plasmids of pcDNA3.4/β2m-linker 2-HLA A24-His tag was successfully constructed and expressed in the Expi293F cells.Conclusion Theβ2m-linker 2-HLA A24 protein has been successfully constructed.And bioinformatics results show that the protein has the function of binding to antigen peptides and activating T cells.It has established a foundation for activating antigen-specific CD8^+T cell stimulated by this protein and peptide in the subsequent experiments.

关 键 词：β2微球蛋白-接头蛋白2-人类白细胞抗原A24(β2m-linker 2-HLA A24) 生物信息学 真核表达 

分 类 号：Q811.4[生物学—生物工程] Q816