摘要
目的:制备链亲和素标记的人白细胞介素24融合蛋白(SA/hIL24),并鉴定其生物学活性。方法:通过RT-RCR扩增hIL-24基因序列,构建pET24a-SA-hIL24和pET21a-hIL24-SA重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达,对表达的融合蛋白采用镍金属螯合(Ni-NTA)层析和凝胶过滤层析纯化、透析复性。MTT法检测融合蛋白对肿瘤细胞的生长抑制作用,流式细胞仪分析融合蛋白对已生物素化的小鼠膀胱癌MB49细胞的锚定修饰效率。结果:成功构建两种融合蛋白的重组表达质粒,在大肠杆菌中实现高效表达,表达量分别占菌体总蛋白的20%和35%,二次纯化后SA/hIL24融合蛋白纯度可达95%以上。SA/hIL24融合蛋白可高效结合至已生物素化的MB49肿瘤细胞表面(表面锚定修饰率大于90%),hIL24-SA融合蛋白可以剂量依赖的方式抑制Hela细胞的生长,而SA-hIL24融合蛋白对Hela细胞没有明显的抑制生长作用。结论:SA/hIL24融合蛋白的制备为hIL-24表面锚定修饰的新型肿瘤细胞疫苗提供了基础。
Objective： To prepare streptavidin-tagged interleukin-24 bifunctional fusion protein （SA/hIL24）and evaluate its biological activity. Methods： The hIL-24 gene was amplified by RT-PCR and inserted into the vector of pET24a-SA and pET2 l a-SA to get pET24a-SA-hIL24 and pET21a-hIL24-SA recombinant plasmids.The SA/hIL24 fusion proteins were expressed in E.coli BL21 （DE3） competent cells and purified by Ni-NTA affinity chromatography and gel filtration chromatography, then refolded by dialysis. MTT method was used toevaluate the inhibitory effect of SA/hIL24 fusion protein. Flow cytometry technology was applied to detect the anchor rate of SA/hIL24 fusion protein to biotinylated MB49 tumor cells. Results： The pET24a-SA-hIL24 andpET21a-hIL24-SA recombinant plasmids were correctly constructed and the SA/hIL24 fusion proteins were expressed in E. coli BL21 （DE3） at about 20%, 35% of total bacterial proteins. The purity of SA/hIL24 was above95% through Ni-NTA affinity chromatography and gel filtration chromatography. SA/hIL24 fusion proteins were efficiently and durably displayed on the surfaces of biotinylated MB49 tumor cells （anchoring modified rate wasabove 90%）. MTT analysis showed that hIL24-SA fusion protein caused a dose-dependent inhibition of Hela cells proliferation, While SA-hIL24 fusion protein couldn＇t inhibit cells proliferation significantly. Conelusion： TheSA/hIL24 fusion protein is expected to be developed a hIL-24-surface-modified cancer cell for the treatment of tumors.
出处
《温州医学院学报》
CAS
2013年第2期78-83,共6页
Journal of Wenzhou Medical College
基金
浙江省重大科技专项(2008C14082)
