PET32a-IL-1RⅠ重组质粒的构建及表达

Construction and expression of recombinant PET32a-IL-1RⅠplasmid

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 recombinant plasmid PET32a-IL-1R I and express extracellular region of human IL-1R I protein in E. coll. Methods Total RNA was extracted from human white blood cells and used as template to amplify the extracellular region of human IL-1R I gene fragments amplified by PCR. Recombinant plasmids were constructed by insert the PCR products into vector plasmid and then were cloned by being transformed into E. coli DHScx. Recombinant plasmid PET32a-IL-1R I was transformed into BL21 （DE3）, the expression of proteins, encoded by recombinant plasmid, was induced with isopropy-β-D-thiogalactoside （IPTG） ,and expressed proteins were identified by SDS PAGE and Western Blot. Results Recombinant plasmid PET32a-IL-1R I , constructed by inserting the extracellular region of IL-1R I gene into vector plasmid, was confirmed by colony PCR and DNA se quencing,and was transformed into expression strain BL21 （DE3）. The expression of prokaryotic protein, with a relative molecular mass about 67 × 10^3 ,induced by IPTG, in transformed BL21 （DE3） was confirmed by SD-PAGE and Western-Blot. Conclusion The extracellular region of IL 1R I gene was successfully cloned and expressed in BL21（DE3） and the elementary expression conditions were obtained,which lays a basis on the further research of IL-1R I .