重组小鼠MIG趋化因子的高效真核表达及免疫活性测定

High-yield production of recombinant mouse monokine induced by interferon-γ in baculovirus expression system and its immunological activity

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 eDNA of mouse CXC chemokine monokine induced by interferon-γ （MIG） and obtain large quantities of purified functional MIG protein using haculovirus expression system. Methods Mouse MIG eDNA was amplified by RT-PCR and cloned into Bat-to-Bat haculovirus expression vector. Recombinant mouse MIG protein （rmMIG） was expressed in High-Five insect cells, and then purified by single-step S-Sepharose from culture medium. The identity of rmMIG was verified by SDS-PAGE and Western blotting. The activity of rmMIG was tested by intracellutlar Ca^2＋ mobilization assay and lymphocytes ehemotaxis assay, Results rmMIG was expressed as secreted protein by insect cells. After single-step S-Sepharose purification, rmMIG was enriched to over 85% of the total protein present in the elution. The yield of rmMIG was 10 mg/L of culture medium, rmMIG protein migrated as several species ranging from 16 000 to 11 000 with 15 000 as the major hand on SDS-PAGE and was confirmed by Western blotting. Purified rmMIG elicited a profound calcium influx in CTLL2 lymphoeytes and induced an enhanced migration of mouse splenocytes in ehemotaxis assay. Conclusion rmMIG can be expressed in haculovirus as secreted protein with high efficiency and can be purified by single-step purification, rmMIG protein retains the expected bioactivity and can be used to facilitate further study of other functions both in vitro and in vivo.