人白细胞介素1受体拮抗基因的克隆及其在大肠杆菌中的高效表达

Cloning and high yield expression of human interleukin 1 receptor antagonist in E.coli

目的 获得一株重组人白细胞介素 1受体拮抗蛋白的高效表达菌株 ,为研究各种急慢性炎症疾病的发病机制以及拮抗 IL - 1的生物学效应奠定基础 .方法 分离人外周血淋巴细胞 ,提取 m RNA,反转录 PCR获得人白细胞介素 1受体拮抗基因 (h IL - 1ra) ,克隆入大肠杆菌表达载体 ,从而构建高效表达人 IL - 1ra的重组菌株 .结果 成功构建了 IL - 1ra的高效表达菌株 (p L DH- h IL 1ra) ,序列测定与文献报道一致 .12 % SDS- PAGE分析显示此菌株所表达的目的蛋白产物相对分子质量为 2 3ku,与预期结果相符 .光密度扫描重组 h IL-1ra蛋白质占细菌总蛋白的 30 % .结论 在大肠杆菌中成功地表达了 h IL- 1ra基因 .

AIM To obtain a high expression clone of human interleukin 1 receptor antagonist (hIL 1ra), which is likely to be of great importance in the pathogenesis of both acute and chronic inflammatory diseases and a competitive inhibitor of the binding of IL 1 to IL 1R. METHODS mRNA wasextracted from human peripheral blood monocytes (PBMC), and the coding sequence of hIL 1ra was amplified by reverse transcription polymerase chain reaction. The expression vector pLDH hHIL1ra was constructed by inserting the PCR products of IL 1ra into the specially designed prokaryotic vector pLDH99. RESULTS By temperature induction, the IL1 ra was highly expressed in E.coli DH5α. 12% SDS PAGE showed that the molecular weight of expression band was about 23 ku, and the expression protein accounted for 30% of total bacterial protein. CONCLUSION Human IL 1ra has effectively been expressed in E.coli .