表皮生长因子受体干扰序列与白细胞介素24融合蛋白的原核表达

目的：在大肠杆菌中表达表皮生长因子受体干扰序列（EGFRi）与白细胞介素24（IL-24）的融合蛋白。方法：人工合成肿瘤细胞表面受体特异性结合位点EGFRi核苷酸序列，应用重叠延伸PCR技术将其与IL-24基因连接，其间引入一段柔软短肽编码基因；将融合基因克隆入pET-22b原核表达载体，IPTG诱导融合蛋白在大肠杆菌BL21（DE3）中表达，对表达条件进行优化；用His·Bind纯化试剂盒对表达产物进行纯化，SDS-PAGE进行鉴定。结果：酶切和测序结果证实EGFRi-IL-24融合基因的原核表达载体构建正确；在IPTG浓度为0.8mol／L、28℃诱导10h的条件下，可溶性融合蛋白表达量最高；表达产物的相对分子质量与预期值一致，为22000；经纯化得到了均一的融合蛋白。结论：获得大肠杆菌表达的融合蛋白EGFRi-IL-2，可用于活性分析。

Prokaryotic Expression of Fusion Protein EGFRi-IL-24

Objective： To obtain fusion protein EGFRi-IL-24 expressed in E.coli. Methods： Gene of epithelial growth factor receptor interference sequence （EGFRi） which was included in primers was linked up with gene of interleukin 24 （IL-24） by overlapping PCR. The fusion fragment was inserted into the prokaryotic vector pET-22b to construct the expression plasmid, and then it was transformed into E.coli BL21（DE3）. The fusion protein was expressed after induction of IPTG, and it was purified with His. Bind purification kit. Results： Restriction enzyme digestion analysis and DNA sequencing showed that EGFRi-IL-24 fusion gene was constructed successfully. When induced by 0.8 mol/L IPTG lasted 10 h at 28℃, majority of fused protein was observed on SDS-PAGE in soluble form. SDS-PAGE showed that fusion protein was highly expressed with the Mr of 22 kD and it was easily to be purified. Conclusion： Fusion protein EGFRi-IL-24 was obtained, which is await for further functional assay.