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目的对HIV-1辅助受体趋化因子受体5(CCR5)配体人类CC3配体样蛋白1 (CCL3L1)进行果蝇Schneider-2细胞融合蛋白表达,纯化后活性分析。方法克隆人类CCL3L1 cDNA,构建CCL3L1表达载体pMT/BiP/His,获得Schneider-2果蝇细胞表达的His-CCL3L1融合蛋白,同时克隆pCDNA3.1-flag-CCR5表达载体,培养稳定表达flag-CCR5的细胞株,检测表达人趋化因子CCL3L1活性。结果成功构建人趋化因子CCL3L1融合蛋白真核表达载体pMT/BiP/His,表达并纯化出融合蛋白His-CCL3L1。免疫沉淀法和Western印迹法分析发现,纯化的His-CCL3L1蛋白能特异性结合CCR5受体,His-CCL3L1蛋白在浓度1～50 nmol/L存在剂量依赖性,50～100 nmol/L无剂量依赖性。结论果蝇Schneider-2细胞表达的His-CCL3L1蛋白具有与天然CCL3L1相同的生物学活性,为进一步探讨CCL3L1影响HIV-1感染的机制奠定了基础。
Objective To analyze the expression of Schneider-2 cell fusion protein of human CC3 ligand-like protein 1 (CCL3L1), a chemokine receptor 5 (CCR5) ligand of HIV-1, and analyze its activity after purification. Methods Human CCL3L1 cDNA was cloned and the CCL3L1 expression vector pMT / BiP / His was constructed. The His-CCL3L1 fusion protein was expressed in Schneider-2 Drosophila cells and the pCDNA3.1-flag-CCR5 expression vector was cloned. Cell lines were tested for expression of human chemokine CCL3L1 activity. Results The eukaryotic expression vector pMT / BiP / His of human chemokine CCL3L1 fusion protein was successfully constructed and the fusion protein His-CCL3L1 was expressed and purified. Immunoprecipitation and Western blotting analysis showed that the purified His-CCL3L1 protein could specifically bind to CCR5 receptor. His-CCL3L1 protein was dose-dependent at concentrations of 1 ~ 50 nmol / L and no dose-dependent at 50 ~ 100 nmol / L Sex. Conclusion The His-CCL3L1 protein expressed by Drosophila Schneider-2 cells has the same biological activity as native CCL3L1, which lays a foundation for further investigation of the mechanism of CCL3L1 affecting HIV-1 infection.