重组人Tim-3蛋白的制备及基于亲和超滤-液质联用技术的天然配体筛选  

Preparation of recombinant human Tim-3 protein and screening of natural ligands based on affinity ultrafiltration-liquid mass spectrometry

作　　者：杨忠杰 李森森[1,2] 曹娟 应真真 张永威 王瑞 YANG Zhongjie;LI Sensen;CAO Juan;YING Zhenzhen;ZHANG Yongwei;WANG Rui(Luohe Central Hospital,Luohe 462000,China;Medical College of Huanghuai University,Zhumadian 463000,China;Henan Provincial Key Laboratory of Traditional Chinese Medicine Preparations and Processing Chinese Medicine,Luohe 462000,China)

机构地区：[1]漯河市中心医院,河南漯河462000 [2]黄淮学院医学院,河南驻马店463000 [3]河南省中药制剂与加工中医药重点实验室,河南漯河462000

出　　处：《药物评价研究》2023年第3期545-551,共7页Drug Evaluation Research

基　　金：河南省科技攻关项目(212102311110);漯医专〔2021〕207号(2021LYZKJXM034)。

摘　　要：目的构建人T细胞免疫球蛋白黏蛋白-3(Tim-3)-His表达载体,表达纯化Tim-3重组蛋白,建立亲和超滤-液质联用技术筛选Tim-3蛋白天然配体。方法利用DNA重组技术构建人Tim-3-His表达质粒,转染HEK293细胞表达Tim-3重组蛋白,通过Ni柱亲和色谱柱进行蛋白纯化,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析Tim-3重组蛋白纯度。10μL受试物(黄芪甲苷、毛蕊异黄酮葡萄糖苷、白术内酯Ⅰ、芒柄花黄素、毛蕊异黄酮)溶液、180μL磷酸盐缓冲溶液(pH 7.4,50 mmol·L−1)与10μL Tim-3(0.84 mg·mL^(−1))混合,在37°C黑暗孵育1 h后,转移至1×10^(4)超滤离心管中以12000 r·min^(−1)离心10 min,将沉淀加入200μL甲醇-水(90∶10)中室温解离10 min,12000 r·min^(−1)离心10 min,收集滤液,进行UPLC-MS/MS分析。通过比较受试物组和Tim-3蛋白变性组中超滤液中待测物的峰面积,计算各待测物特异结合率。结果经双酶切及测序鉴定证明,人Tim-3-His重组表达质粒构建正确;纯化的人Tim-3重组蛋白质量分数达90%以上;建立的亲和超滤-液质联用筛选体系专属性、精密度、重复性、稳定性良好;白术内酯Ⅰ可与Tim-3蛋白特异性结合。结论成功表达了可溶性、高纯度的人Tim-3重组蛋白,成功建立亲和超滤-液质联用筛选体系,白术内酯Ⅰ可与Tim-3蛋白特异性结合。Objective To construct a prokaryotic expression vector of T cell immunoglobulin mucin-3(Tim-3)-His,express and purify the recombinant Tim-3 protein,and construct an ultrafiltration affinity technology to screen natural ligands of Tim-3 protein.Methods Human Tim-3-His expression plasmid was constructed by DNA recombination technology,and then transfected into HEK293 cells to express Tim-3 recombinant protein.The protein was purified by Ni column affinity chromatography,and the purity of Tim-3 recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).Test substance(astragaloside A,calyx isoflavone glucoside,atractylolactone I,anthocyanin,and calyx isoflavone)solution of 10μL,180μL phosphate buffer solution(pH 7.4,50 mmol·L−1)and 10μL Tim-3(0.84 mg·mL^(−1))mixed,incubated at 37℃for 1 h in dark,transferred to 1×10^(4)centrifuge with 12000 r·min^(−1)in ultrafiltration centrifuge tube for 10 min,and added the sediment to 200μL methanol-water(90∶10)dissociated at room temperature for 10 min,centrifuged 12000 r·min^(−1)for 10 min,and the filtrate was collected for UPLC-MS/MS analysis.The specific binding rate of each analyte was calculated by comparing the peak area of the analyte in the ultrafiltration fluid of the test substance group and the Tim-3 protein denaturation group.Results The recombinant expression plasmid of human Tim-3-His was constructed correctly by double restriction enzyme digestion and sequencing.The mass fraction of purified human Tim-3 recombinant protein is more than 90%.The established affinity ultrafiltration-LC-MS screening system has good specificity,precision,repeatability and stability.Atractylolide I can specifically bind to Tim-3 protein.Conclusion The recombinant expression plasmid of human Tim-3-His was constructed correctly by double restriction enzyme digestion and sequencing.The mass fraction of purified human Tim-3 recombinant protein was more than 90%.The established affinity ultrafiltration-LC-MS screening system had

关 键 词：T细胞免疫球蛋白黏蛋白-3 重组蛋白 亲和超滤-液质联用 白术内酯Ⅰ 

分 类 号：R392.12[医药卫生—免疫学] R285.5[医药卫生—基础医学]