目的 構(gòu)建唾液酸免疫球蛋白凝集素1（Siglec-1）原核表達(dá)載體，表達(dá)純化Siglec-1重組蛋白，構(gòu)建超濾親和-液質(zhì)聯(lián)用技術(shù)（UF-LC-MS）篩選 Siglec-1蛋白天然配體。方法 利用 DNA重組技術(shù)構(gòu)建 Siglec-1重組蛋白原核表達(dá)載體，轉(zhuǎn)化到BL21（DE3）感受態(tài)細(xì)胞中表達(dá)Siglec-1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（SDS-PAGE）電泳分析Siglec-1重組蛋白純度。建立UF-LC-MS技術(shù)研究金銀花、甘草、迷迭香、菊苣中6個(gè)主要組分綠原酸、迷迭香酸、阿魏酸、甘草酸、甘草次酸、菊苣酸對(duì) Siglec-1蛋白的親和力，通過(guò)比較樣品組和蛋白變性組超濾液中待測(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，重組表達(dá)質(zhì)粒 pET-22b-Siglec-1-His構(gòu)建正確；純化的人Siglec-1重組蛋白質(zhì)量分?jǐn)?shù)達(dá)90%以上；建立了UF-LC-MS篩選體系，篩選出甘草次酸可與Siglec-1蛋白特異性結(jié)合。結(jié)論 成功表達(dá)了高純度、高產(chǎn)量的人Siglec-1重組蛋白，篩選出甘草次酸可與Siglec-1蛋白特異性結(jié)合。

Objective To construct a prokaryotic expression vector of sialoadhesin (Siglec-1), express and purify the recombinant Siglec-1 protein, and construct an ultra-filtration affinity-liquid chromatography-mass spectrometry (UF-LC-MS) technique to screen natural ligands of Siglec-1 protein.Methods The prokaryotic expression vector of Siglec-1 recombinant protein was constructed by DNA recombination technology. The Siglec-1 recombinant protein was expressed and purified, the purity of Siglec-1 recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein was purified by Ni column affinity chromatography. An UF-LC-MS technique was established to screen the affinity of six main components (chlorogenic acid, 2-rosmarinic acid, 3-glycyrrhizic acid, 4-chicoric acid, 5-glycyrrhetinic acid, and 6-ferulic acid) in Lonicera japonica, Glycyrrhiza uralensis, Rosmarinus officinalis and Cichorium intybus to Siglec-1 protein. Calculate the specific binding rates of each analyte by comparing the peak areas of the analyte in the ultrafiltration solution of the sample group and the protein denaturation group.Results The double-enzyme digestion and sequencing identification proved that the recombinant expression plasmid pET-22b-Siglec-1-His was constructed correctly, the relative purity of purified human Siglec-1 recombinant protein was over 90%, the UF-LC-MS screening system was established, and glycyrrhetinic acid was screened out. Glycyrrhetinic acid can specifically bind to Siglec-1 protein.Conclusion A high-purity and recombinant human Siglec-1 protein with certain chemotactic activity was successfully expressed. It was screened out that glycyrrhetinic acid can specifically bind to Siglec-1 protein.

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國(guó)家自然科學(xué)基金資助項(xiàng)目（82204727）；河南省科技公共項(xiàng)目（242102310566）