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。10 μL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、180 μL磷酸盐缓冲溶液（pH 7.4，50 mmol·L^-1）与10 μL Tim-3（0.84 mg·mL^-1）混合，在37℃黑暗孵育1 h后，转移至1×10⁴超滤离心管中以12 000 r·min^-1离心10 min ，将沉淀加入200 μL甲醇-水（90∶10）中室温解离10 min，12 000 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 10 μL, 180 μL phosphate buffer solution (pH 7.4, 50 mmol·L^-1) and 10 μL Tim-3 (0.84 mg·mL^-1) mixed, incubated at 37 ℃ for 1 h in dark, transferred to 1×10⁴ centrifuge with 12 000 r·min^-1 in ultrafiltration centrifuge tube for 10 min, and added the sediment to 200 μL methanol-water (90∶ 10) dissociated at room temperature for 10 min, centrifuged 12 000 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 good specificity, precision, repeatability and stability. Atractylolide I can specifically bind to Tim-3 protein.

R392.12;R285.5

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