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高志昕应用钙盐沉淀法将ｈＩＬ一８重组质粒ＰＨＮＰ１０１转化感受态大肠杆菌ＨＢ１０１，转化菌经扩增培养，其包涵体及胞浆内均发现有重组蛋日的表达（约１０：１）。表达蛋白经初步分离，肝素柱、离子交换柱和凝胶柱进一步纯化，获得了高纯度的ｒｈＩＬ—８（＞９５％）。纯化的中ＩＬ—８在新西兰免体内白细胞动员试验中显示了较高的生物学活性硕士研究生＊导师脉采血，计数其外周血白细胞总数。对照组静脉注射等体积灭菌生理盐木，并于相同的时间点干血检查。２结果２．１重组质拉ｐＨＮＰ１０１转化ＨＢ１０１细菌：转化菌可在含１００ｍｇ／Ｌ氨苄青霉素的琼脂平板上生长并形成定落．空日质粒转化细菌则不能生长。２．２纯化ｒｈＩＬ——８的ＳＤＳ—ＰＡＧＥ：成熟ｈＩＬ——８是一低分子蛋曰，分子量约为１０ｋｕ．初步分离的ｒｈＩＬ——８经纯化后其纯度逐步提高（＞９５％），于电泳图谱上１０ｋｕ处显示单一条带（附图）。２．３纯化ｒｈＩＬ——８的ＥＬＩＳＡ检测；ｈＩＬ——８ＥＬＩＳＡ检测结果显示上述纯化后的蛋白为特异性ｒｈＩＬ——８，其含量约为２５ｍｇ／Ｌ。２．４ｒｈＩＬ——８快速募集白细胞进人外周血循环：在静注，ｒｈＩＬ——８（１０μｇ／ｋｇ）５ｍｉｎ后，兔外
GAO Zhi-Xin The hIL-8 recombinant plasmid PHNP101 was transformed into competent E. coli HB101 by calcium salt precipitation method. The transformed bacteria were expanded and cultured. The expression of recombinant protein was found in inclusion bodies and cytoplasm (about 10: 1). The expressed protein was separated and purified by heparin column, ion exchange column and gel column. High purity rhIL-8 (> 95%) was obtained. Purified IL-8 showed high biological activity in New Zealand white blood cells-free in vitro mobilization master’s program * Tutor blood was collected and the total number of peripheral white blood cells was counted. Control group intravenous injection of equal volume of physiological saline, and at the same time dry blood examination. 2 Results 2.1 Recombinant Plasmid pHNP101 Transformation of HB101 Bacteria: Transformants can grow and colonize on agar plates containing 100 mg / L ampicillin. Air-day plasmid-transformed bacteria can not grow. 2.2 Purification of rhIL - 8 by SDS-PAGE: Mature hIL - 8 is a low molecular weight egg said molecular weight of about 10ku. The purity of the initially isolated rhIL - 8 was gradually increased (> 95%), showing a single band at 10ku on the electrophoresis map (with photos). 2.3 ELISA assay of purified rhIL - 8; hIL - 8 ELISA test results showed that the purified protein was specific rhIL - 8, its content was about 25mg / L. Rapid recruitment of leukocytes into the peripheral blood of 2.4 rhIL - 8: After intravenous injection of rhIL - 8 (10μg / kg) for 5 minutes,