人可溶性白细胞介素-6受体基因在昆虫细胞内的克隆与表达

摘要

Objective To clone the human soluble interleukin-6 receptor(hsIL-6R) gene and expression in insect cell line. Methods The hsIL-6R gene was cloned into plasmid pAcGP67B. After co-transfection of recombinant plasmid and wild type AcNPV DNA, the rAcNPV was confirmed by the end point dilution assay and dot blot. Then it was purified by plaque assay. Results SDS-PAGE showed molecular weight of the expressed product was about 47 000. The expressed recombinant protein was confirmed to be specific and capable of binding its ligand IL-6 by Western blot and ligand-receptor binding assays. Conclusions Secretory expression of hsIL-6R gene in baculovirus expression system was indicated. The expressed product had immunological and biological activities.%在昆虫细胞中表达人可溶性白细胞介素-6受体(sIL-6R)基因。方法将人sIL-6R cDNA克隆至杆状病毒转移载体pAcGP67B中，再将重组转移载体质粒与野生病毒AcNPV DNA共转染昆虫细胞Sf9；经同源重组后，以终点稀释和斑点杂交法筛选重组杆状病毒；采用空斑分析法纯化重组的病毒，然后以纯化的重组病毒感染昆虫细胞Sf9。结果斑点杂交实验证实，纯化得到的重组病毒含有人sIL-6R基因；SDS-PAGE结果显示，表达产物sIL-6R相对分子质量为47 000左右；Western blot和受体配基结合实验表明，表达产物具有与其配基特异性地结合的能力。结论杆状病毒-昆虫细胞系统能分泌表达sIL-6R，表达产物具有免疫活性和生物活性。