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目的探索 GPI-锚固蛋白的体外表达 ,获得具有生物学活性的可溶性游离 CD5 9分子。方法通过 PCR选择性扩增编码成熟 CD5 9氨基酸序列的基因片段 ,并将之克隆入 Pin Point Xa- 3质粒中。 IPTG诱导融合蛋白表达 ,产物经亲和素树脂亲和层析。在体外微量反应性溶血抑制实验中检测重组 CD5 9纯化样品的生物学活性。结果筛选得到的重组子诱导后表达分子量约为 2 2 k D的生物素化 CD5 9融合蛋白。亲和素树脂纯化得到高纯度的蛋白样品。纯化分子在反应性溶血实验中表现出溶血抑制活性。结论本研究对体外表达无 GPI-锚的可溶性游离 CD5 9分子进行了表达探索 ,获得具一定生物学活性的游离重组 CD5 9,为进一步研制应用型 CD5 9活性分子打下基础。
Objective To explore the expression of GPI-anchored protein in vitro and to obtain a soluble free CD5 9 molecule with biological activity. Methods The gene fragment encoding the mature CD59 amino acid sequence was selectively amplified by PCR and cloned into the Pin Point Xa-3 plasmid. IPTG induced fusion protein expression, the product affinity chromatography with avidin resin. The biological activity of recombinant CD5 9 purified samples was tested in an in vitro microreactivity hemolysis inhibition assay. Results The recombinant plasmid was induced to express biotinylated CD59 fusion protein with molecular weight of about 2 2 kD. Avidin resin purification to obtain high-purity protein samples. Purified molecules show hemolysis inhibitory activity in reactive hemolysis experiments. Conclusions This study explored the expression of soluble free CD5 9 without GPI-anchors in vitro and obtained free recombinant CD5 9 with certain biological activity, which laid the foundation for the further development of active CD5 9 molecule.