抗人IL-1β单链抗体的构建及其中和活性的检测  

Construction and neutralizing activity of anti-hIL-1β scFv

作　　者：李天鹤[1] 任桂萍[1] 徐黎明[2] 周兵[1] 郭茉[1] 齐剑英[1] 张宇[1] 赵景壮[1] 于引航[1] 尹杰超[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 [2]中国水产科学研究院黑龙江水产研究所,哈尔滨1510000

出　　处：《中国免疫学杂志》2013年第3期304-308,311,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金青年科学基金项目(31001084);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);哈尔滨市科技创新人才发展计划资助(2006RFXXS002);东北农业大学创新团队发展计划资助(2007年)

摘　　要：目的:原核表达抗人IL-1β(hIL-1β)的scFv,并对纯化后抗体的特异性、亲和力以及中和活性进行检测,为研制治疗类风湿关节炎的小分子抗体药物奠定基础。方法:使用PCR方法从含有抗hIL-1β抗体基因的质粒中获得抗hIL-1β的单链抗体基因,将其插入原核表达载体pET-27b(+)中,构建重组表达载体pET-27b-A-hIL-1β-scFv。将该载体转化大肠杆菌Rosetta后诱导表达,经包涵体变性、复性得到可溶的抗hIL-1βscFv蛋白。利用高效液相色谱仪对其纯度进行分析后使用Western blot和ELISA方法测定scFv抗体对抗原特异性结合能力和亲和力,使用MTT的方法检测scFv抗体中和人IL-1β蛋白的能力。结果:成功地对抗hIL-1β单链抗体进行构建、诱导、表达和纯化。得到纯度为72.05%、相对分子质量约为28 ku的目的蛋白。ELISA和Western blot结果证实scFv蛋白对hIL-1β具有特异性结合能力。MTT实验结果证实该scFv抗体可以有效阻断hIL-1β刺激L929细胞的增殖。结论:通过原核表达系统得到的抗hIL-1β单链抗体,纯化后,鉴定其与hIL-1β蛋白有特异性结合能力,并且表现出中和hIL-1β的活性,为进一步研究治疗RA的小分子抗体奠定了基础。Objective：To prokaryotically express the scFv against human IL-1 β and test the specificity, affinity and neutrali- zing activity of the purified antibody, and hence to lay the foundation for further development of the small molecular antibody drug for treatment RA. Methods： Anti-hIL-1β single chain antibody gene from existing plasmid （contain anti-h IL-1β antibody gene） was sub- cloned into the prokaryotic expression vector pET-27b to form the expression vector pET-27b-A-hIL-1 β scFv. The construct was trans- formed into Rosetta for expression the target protein. The soluble anti-hIL-1β scFv antibody was obtained through inclusion body denat- uration and renaturation. The antibody specificity and affinity were examined by ELISA and Western blot assay. Ability of the scFv an- tibody to neutralize hIL-1β was tested by MTT assay. Results：Have successfully constructed, expressed and purified the anti-hIL-1 β single chain antibody. The molecular mass of the antibody is about 28 ku. The ELISA and Western blot results confirmed that scFv had specific binding ability with hIL-1 β. The MTT results proved that scFv could effectively block proliferation of L929 cells stimulated by hlL-113. Conclusion：The anti-human hIL-1β single-chain antibody expressed from prokaryotic expression system demonstrates specific- ity and affinity with hIL-1 βand shows the neutralization activity to hIL-1 β. The study lays the foundation for further development of better therapeutic antibodies for treatment of RA.

关 键 词：hIL-1β 单链抗体 包涵体复性 中和活性 MTT 

分 类 号：R392.4[医药卫生—免疫学]