目的 構(gòu)建人T細(xì)胞免疫球蛋白黏蛋白-3（Tim-3）-His表達(dá)載體，表達(dá)純化Tim-3重組蛋白，建立親和超濾-液質(zhì)聯(lián)用技術(shù)篩選Tim-3蛋白天然配體。方法 利用DNA重組技術(shù)構(gòu)建人Tim-3-His表達(dá)質(zhì)粒，轉(zhuǎn)染HEK293細(xì)胞表達(dá)Tim-3重組蛋白，通過(guò)Ni柱親和色譜柱進(jìn)行蛋白純化，十二烷基硫酸鈉聚丙烯酰胺凝膠電泳（SDS-PAGE）分析Tim-3重組蛋白純度。10 μL受試物（黃芪甲苷、毛蕊異黃酮葡萄糖苷、白術(shù)內(nèi)酯Ⅰ、芒柄花黃素、毛蕊異黃酮）溶液、180 μL磷酸鹽緩沖溶液（pH 7.4，50 mmol·L^-1）與10 μL Tim-3（0.84 mg·mL^-1）混合，在37℃黑暗孵育1 h后，轉(zhuǎn)移至1×10⁴超濾離心管中以12 000 r·min^-1離心10 min ，將沉淀加入200 μL甲醇-水（90∶10）中室溫解離10 min，12 000 r·min^-1離心10 min，收集濾液，進(jìn)行UPLC-MS/MS分析。通過(guò)比較受試物組和Tim-3蛋白變性組中超濾液中待測(cè)物的峰面積，計(jì)算各待測(cè)物特異結(jié)合率。結(jié)果 經(jīng)雙酶切及測(cè)序鑒定證明，人Tim-3-His重組表達(dá)質(zhì)粒構(gòu)建正確；純化的人Tim-3重組蛋白質(zhì)量分?jǐn)?shù)達(dá)90%以上；建立的親和超濾-液質(zhì)聯(lián)用篩選體系專(zhuān)屬性、精密度、重復(fù)性、穩(wěn)定性良好；白術(shù)內(nèi)酯Ⅰ可與Tim-3蛋白特異性結(jié)合。結(jié)論 成功表達(dá)了可溶性、高純度的人Tim-3重組蛋白，成功建立親和超濾-液質(zhì)聯(lián)用篩選體系，白術(shù)內(nèi)酯Ⅰ可與Tim-3蛋白特異性結(jié)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

河南省科技攻關(guān)項(xiàng)目（212102311110）；漯醫(yī)專(zhuān)〔2021〕207號(hào)（2021LYZKJXM034）