SA/mIL15双功能融合蛋白的制备及其生物学鉴定  

Preparation and bioactivity evaluation of SA-mIL15 fusing protein

作　　者：陈艳丽[1] 许晓玲[1] 唐佳[1] 聂小霞[1] 宋志纯[1] 胡志明[2] 高基民[1,2] 

机构地区：[1]温州医学院浙江省医学遗传学重点实验室,浙江温州325035 [2]南方医科大学生物技术学院生物治疗研究所,广东广州510515

出　　处：《细胞与分子免疫学杂志》2010年第2期107-110,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助项目(2006AA02Z4C4);浙江省自然科学基金资助项目(R2080407);浙江省科技计划重大专项资助项目(2008C14082)

摘　　要：目的:制备链亲和素(SA)标记的鼠白细胞介素-15融合蛋白SA-mIL15和mIL15-SA,并研究其生物学功能。方法:构建pET24a-SA-L-mIL15和pET21a-mIL15-L-SA重组表达质粒,在大肠杆菌中表达融合蛋白SA-mIL15和mIL15-SA,对所表达的蛋白分别采用镍金属螯合(Ni-NTA)层析和阴离子交换(DEAE)层析进行纯化,透析复性。MTT法检测融合蛋白对ConA刺激的小鼠脾淋巴细胞增殖活性,流式细胞术(FCM)分析融合蛋白对生物素化的小鼠前列腺癌(RM-1)细胞表面锚定修饰效率。结果:SA-mIL15和mIL15-SA在大肠杆菌中实现了高效表达,约占细菌总蛋白的20%。制备的SA-mIL15和mIL15-SA融合蛋白纯度达到95%,并具有双重活性,即:mIL15促进ConA激活的小鼠脾淋巴细胞的增殖活性和SA介导的高效结合至表面已生物素化的RM-1细胞的功能(表面锚定修饰效率均大于95%)。其中,SA-mIL15双功能融合蛋白促进ConA激活的小鼠脾淋巴细胞增殖活性为1×106IU/mg,mIL15-SA活性为2×105IU/mg。结论:SA/mIL15双功能融合蛋白具有双重活性,为mIL15表面锚定修饰的肿瘤细胞疫苗的研制奠定了基础。AIM：To prepare and characterize streptavidin-tagged murine interleukin-15 fusion proteins.METHODS：pET24a-SA-L-mIL15 and pET21a-mIL15-L-SA plasmids were constructed and expressed in Rosetta（DE3） host bacteria to generate SA/mIL15 fusion proteins.SA-mIL15 fusion protein was purified through the Ni-NTA affinity chromatography,and mIL15-SA fusion protein through anion exchange chromatography,followed by refolding.The efficiency of surface modification of the fusion proteins on the biotinylated RM-1 tumor cells was evaluated by a flow cytometer.MTT method was used to evaluate the proliferating effect of SA/mIL15 fusion proteins on mouse spleen lymphocytes stimulated by ConA.RESULTS：Both SA-mIL15 and mIL15-SA fusion proteins were highly expressed in Rosetta（DE3） at about 20% of total bacterial proteins.They exhibited the bi-functionality：proliferation-promoting activity of mIL15 on mouse spleen lymphocytes with the specific activity of 1×106 IU/mg for SA-mIL15 or 2×105 IU/mg for mIL15-SA,and SA-mediated high-affinity binding to the biotinylated surfaces of RM-1 tumor cells with about 95% surface modification efficiency.CONCLUSION：SA/mIL15 bi-functional fusion proteins were generated,which made feasible the development of mIL15-surtface-modified cancer cell vaccine.

关 键 词：白细胞介素15 链亲和素 融合蛋白 锚定 

分 类 号：R979.1[医药卫生—药品] R282.71[医药卫生—药学]