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目的克隆小鼠CXC趋化因子干扰素-γ诱生单核因子(MIG)并在杆状病毒表达系统中进行高效表达,进而对MIG蛋白进行纯化和活性鉴定。方法从小鼠脑组织中用RT-PCR的方法扩增出小鼠MIG基因的全长cDNA,在克隆入Bac-to-Bac杆状病毒表达载体后在High-Five昆虫细胞中进行分泌型表达;表达产物经S-Sepharose进行了纯化并进行了生化鉴定;最后对纯化的重组小鼠MIG蛋白进行了钙离子内流诱发试验和淋巴细胞趋化测定以鉴定其免疫活性。结果成功克隆了小鼠MIG基因并在Bac-to-Bac杆状病毒表达系统中进行了高效表达,其表达蛋白主体相对分子质量约为15 000;经过单步S-Sepha-rose纯化法就可获得纯度为85%以上的重组小鼠MIG蛋白,产量为10 mg/L;该蛋白该纯化蛋白在体外具有诱导小鼠T淋巴细胞钙离子内流和吸引、趋化小鼠脾细胞的活性。结论小鼠MIG蛋白可在杆状病毒表达系统中高产量分泌表达并可用S-Sepharose纯化法直接纯化;小鼠重组MIG蛋白具有激活、趋化淋巴细胞的生物活性;rmMIG纯化蛋白的大量制备为进一步研究小鼠MIG蛋白在炎症、免疫器官的成熟和其它疾病中的功能和作用提供了有利的工具。
Objective To clone the mouse CXC chemokine interferon-γ-induced monocyte-macrophage (MIG) gene and express it in the baculovirus expression system for further purification and identification of the MIG protein. Methods The full length cDNA of mouse MIG gene was amplified by RT-PCR from mouse brain. The full length cDNA of mouse MIG gene was cloned into Bac-to-Bac baculovirus expression vector and secreted in High-Five insect cells. The expressed product was purified by S-Sepharose and biochemically identified. Finally, the purified recombinant mouse MIG protein was assayed for calcium influx and lymphocyte chemotaxis assay to identify its immunological activity. Results The mouse MIG gene was successfully cloned and expressed in Bac-to-Bac baculovirus expression system. The relative molecular mass of the expressed protein was about 15 000. After single-step S-Sepharose purification The recombinant mouse MIG protein with a purity of over 85% was obtained with a yield of 10 mg / L. The purified protein had the activity of inducing influx and attraction of mouse T lymphocytes in vitro and chemotaxis to mouse splenocytes. Conclusion Mouse MIG protein can be expressed in high yield in baculovirus expression system and can be directly purified by S-Sepharose purification. Mouse recombinant MIG protein has the activity of activating and chemotactic lymphocyte. The preparation of rmMIG purified protein is as follows It is an advantageous tool to study the function and role of mouse MIG protein in inflammation, maturation of immune organs and other diseases.