抗IL-1βscfv抗体与TNF—α可溶性受体融合蛋白的构建及其生物学活性分析  

Construction, expression, and bio-activity assay of an anti-IL-1βcfv and TNFR1 fusion protein

作　　者：阚方明[1] 任桂萍[1] 郭茉[1] 韩阳[1] 齐剑英[1] 张宇[1] 张雅坤[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院生物制药教研室,哈尔滨150030

出　　处：《中华微生物学和免疫学杂志》2012年第10期855-860,共6页Chinese Journal of Microbiology and Immunology

基　　金：黑龙江省发改委项目（[2011]1570）;黑龙江省博士后科研启动基金项目（LBH-Q09162）;黑龙江省教育厅科学技术研究项目（12521z004）

摘　　要：目的设计原核表达抗人白细胞介素．1B单链抗体（IL-1βscfv）与TNF．仪可溶性受体的融合蛋白，并分析其生物学活性，为类风湿关节炎的治疗提供新的候选药物。方法利用RT-PCR方法从HeLa细胞总RNA中扩增了人肿瘤坏死因子I型受体胞外区基因片段，与抗人IL-1βscfv通过抗体铰链区融合，克隆至pET27b（＋）表达载体中，构建成pET-IL-1scfv：TNFR1质粒；转化大肠杆菌Rosetta进行表达。从包涵体中纯化得到IL-1scfv：TNFR1蛋白，进行IL-1scfv：TNFR1的鉴定及活性检测。结果ELISA结果证明，IL-1scfv：TNFRl可以分别结合hIL-1β和hTNF-α，并呈现剂量依赖性，表明IL-1scfv：TNFR1的单链抗体部分和可溶性受体部分可以各自形成正确的空间构象；免疫斑点分析证明，IL-1scfv：TNFRl与hTNF—α结合后仍可以结合hIL-1β，具有同时结合hlL-1β和hTNF-α的两种靶分子的能力，表明IL-1scfv：TNFR1的两个部分不会相互影响与靶分子的结合。活性测定结果表明，IL-1scfv：TNFRl可以有效封闭肿瘤坏死因子对L929细胞的细胞毒效应，具有显著抑制hlL-1β促进L929细胞增殖的活性，IL-1scfv：TNFR1对两种靶分子的拮抗作用均呈现明显的剂量依赖性。结论成功构建了能够同时结合hIL-1β和hTNF-α两种靶因子的双特异性IL-1scfv：TNFR1融合蛋白，并能有效抑制hTNF-α和IL-1β的生物活性，为类风湿关节炎的治疗提供新的候选药物。Objective To express the anti-IL-1βscfv and soluble TNF receptor 1 （sTNFR1）, and analyze their bio-activities. Methods sTNFR1 was obtained by RT-PCR from the total RNA of HeLa cells, and fused with IL-1βscfv by the hinge fragment of IgG molecule. The fusion gene IL-1 scfv： TNFR1 was cloned into the expression vector pET27b（＋）. The fusion protein was expressed and purified from inclusion bodies. Results The ELISA analysis showed that the fusion protein could bind hIL-1β and hTNF-α respectively in a dose-dependent manner, indicating that scfv and sTNFR in the fusion protein can form the correct spatial configuration. The dolt-blot analysis showed that the fusion protein could concurrently bind with hiL-1β and hTNF-α, indicating that the combination of the two parts of the fusion protein does not influence each other for binding to their target molecules. The bioactivity assay showed that the fusion protein could inhibit both the cytotoxicity of hTNF-α on L929 cells and hIL-1 G-induced proliferation of L929 cells, indicating that the fusion protein has the ability to neutralize hTNF-α and hIL-1β. Conclusion A bispecific fusion protein IL-1 scfv：TNFR1 was successfully constructed. The fusion protein has the ability to inhibit the biological activity of hTNF-α and hIL-1β, and provides a drug candidate for the treatment of rheumatoid arthritis.

关 键 词：白细胞介素1Β 肿瘤坏死因子Α 可溶性肿瘤坏死因子受体1 类风湿关节炎 

分 类 号：R392[医药卫生—免疫学]