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目的构建含有编码白细胞介素1Ⅰ型受体(IL-1R I)胞外区蛋白基因的PET32a-IL-1RⅠ重组质粒,利用原核表达体系表达IL-1RⅠ的胞外区蛋白。方法从人白细胞中提取总RNA,采用聚合酶链反应(PCR)从总RNA中扩增出IL-1RⅠ胞外段基因,并将其插入PET32a质粒,构建重组质粒,化学法转化大肠杆菌DH5α进行克隆。将克隆得到的PET32a-IL-1RⅠ重组质粒转化入表达菌株BL21(DE3),通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导其蛋白表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western-Blot鉴定蛋白表达效果。结果菌落PCR及DNA测序证实IL-1RⅠ胞外段基因已正确克隆到载体中;重组质粒成功转入表达菌株BL21(DE3),SDS-PAGE和Western-Blot结果显示表达菌经IPTG诱导后表达出相对分子质量为67×103左右的蛋白。结论成功地克隆了人IL-1RⅠ胞外段基因并在大肠杆菌中进行了表达,为IL-1RⅠ的进一步研究打下了基础。
OBJECTIVE: To construct the recombinant plasmid PET32a-IL-1RⅠ containing the gene encoding the extracellular region of IL-1R I and to express the extracellular domain of IL-1RI using prokaryotic expression system. Methods Total RNA was extracted from human leukocytes. The extracellular domain of IL-1RⅠ gene was amplified from total RNA by polymerase chain reaction (PCR) and inserted into PET32a plasmid to construct a recombinant plasmid. The recombinant plasmid was transformed into E. coli DH5α clone. The cloned PET32a-IL-1RⅠ recombinant plasmid was transformed into the expression strain BL21 (DE3) and its protein expression was induced by isopropyl-β-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis -PAGE) and Western-Blot identification of protein expression. Results The colony PCR and DNA sequencing confirmed that the extracellular domain of IL-1RI was correctly cloned into the vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) successfully. SDS-PAGE and Western-Blot showed that the recombinant plasmid was induced by IPTG Relative molecular mass of about 67 × 103 protein. Conclusion The human IL-1RⅠ extracellular domain gene was successfully cloned and expressed in E. coli, which laid the foundation for the further study of IL-1RⅠ.