重组人Siglec-1蛋白的制备及基于超滤亲和-液质联用技术天然配体的筛选

Preparation of recombinant human Siglec-1 protein and screening of natural ligands based on affinity ultrafiltration-liquid mass spectrometry

目的构建唾液酸免疫球蛋白凝集素1(Siglec-1)原核表达载体,表达纯化Siglec-1重组蛋白,构建超滤亲和-液质联用技术(UF-LC-MS)筛选Siglec-1蛋白天然配体。方法利用DNA重组技术构建Siglec-1重组蛋白原核表达载体,转化到BL21(DE3)感受态细胞中表达Siglec-1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(SDS-PAGE)电泳分析Siglec-1重组蛋白纯度。建立UF-LC-MS技术研究金银花、甘草、迷迭香、菊苣中6个主要组分绿原酸、迷迭香酸、阿魏酸、甘草酸、甘草次酸、菊苣酸对Siglec-1蛋白的亲和力,通过比较样品组和蛋白变性组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,重组表达质粒pET-22b-Siglec-1-His构建正确;纯化的人Siglec-1重组蛋白质量分数达90%以上;建立了UF-LC-MS筛选体系,筛选出甘草次酸可与Siglec-1蛋白特异性结合。结论成功表达了高纯度、高产量的人Siglec-1重组蛋白,筛选出甘草次酸可与Siglec-1蛋白特异性结合。

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.