小鼠IL-35基因的克隆及在大肠杆菌中的表达

摘要

目的 克隆小鼠白细胞介素-35(IL-35)基因并在大肠杆菌中表达和纯化.方法 用RT-PCR方法从小鼠脾脏扩增出IL-35的两个亚基Ebi3和p35的成熟肽基因片段,采用重叠PCR法将Ebi3基因和p35基因通过(Gly4Ser)3接头连接,得到的融合基因片段先克隆于pMD19-T载体,再亚克隆到表达载体pET-30b(+),并转化大肠杆菌BL21(DE3),用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,Western blot检测目的蛋白,用Ni螯合层析方法纯化表达产物.结果 从小鼠脾细胞总RNA中扩增出Ebi3和p35的基因片段,重叠PCR法得到的融合基因经鉴定含预期序列；构建的重组表达载体pET-30b(+)-IL-35在BL21(DE3)中经IPTG诱导后表达出约50kd的融合蛋白；Western blot检测结果显示,该融合蛋白能与抗6×His的单克隆抗体发生反应；经Ni螯合层析方法纯化获得单一蛋白条带.结论 小鼠IL-35在大肠杆菌中表达并纯化,为进一步研究其功能及应用价值奠定了良好的基础.%Objective To express IL-35 gene in E. coli and purify IL-35 protein. Methods Mouse Ebi3 and p35 mature peptide gene were amplified by RT-PCR from mouse spleen. By overlap PCR, both genes were fused with the ( Gly4Ser)3linker. The fusion gene was inserted initially into pMD19-T vector, then it was subcloned into the expression vector pET-30b ( + ). and was transformed into E. coli BL21 (DE3). The target protein was induced by IPTG and detected by anti-6 x His monoclonal antibody and purified by affinity chromatography. Results Cloned mouse IL-35 gene was identified to contain the expected sequence, the target gene was expressed in the form of fusion protein with relative molecular weight at 50 kd. The protein can be recognized by anti-6 x His monoclonal antibody and can be purified by affinity chromatography. Conclusion Mouse IL-35 was expressed and purified successfully, which lays the foundation for studying the functions and application value of mouse IL-35.