小鼠AChR(m97-116)肽段-CD205融合单链抗体的表达及活性检测  

Determining the expression and affinity of mice acetylcholine receptor peptide 97-116and CD205fusion single chain antibody fragment

作　　者：孙超[1] 袁媛[2] 郑鹏飞[3] 高星[2] 赵元琳 李乐[2] 常婷[1] 李柱一[1] 

机构地区：[1]陕西省西安市第四军医大学唐都医院神经内科,710038 [2]第四军医大学病理学教研室,710032 [3]第四军医大学西京医院心脏内科,710032

出　　处：《中国神经免疫学和神经病学杂志》2015年第1期4-8,共5页Chinese Journal of Neuroimmunology and Neurology

基　　金：国家自然科学基金资助项目(81102217)

摘　　要：目的构建小鼠AChR(m97-116)肽段-CD205融合单链抗体原核表达载体,表达融合单链抗体(m97-116-scFv205),检测其对未成熟树突状细胞(iDC)的亲和力,为通过iDC诱导AChR特异性免疫耐受提供实验基础。方法分离培养小鼠原代骨髓祖细胞,经粒细胞-巨噬细胞集落刺激因子(GM-CSF)10ng/mL和白细胞介素4(IL-4)10ng/mL刺激培养,经脂多糖(LPS)1ng/mL诱导24h后,高倍显微镜及扫描电镜观察细胞形态;流式细胞术(FCM)检测细胞表面相关分子表达,对iDC表型进行鉴定;构建原核表达载体m97-116-scFv205-pET22b(+),0.1 mmol/mL异丙基硫代半乳糖苷(IPTG)18℃诱导表达18h,镍(Ni)柱纯化,SDSPAGE和Western blot检测融合单链抗体m97-116-scFv205表达及纯化;FCM检测其与iDC的亲和力。结果形态学观察提示培养细胞为DC,FCM结果显示培养细胞符合iDC表型;SDS-PAGE和Western blot在上清中检测到m97-116-scFv205可溶性表达;亲和力检测显示m97-116-scFv205与iDC具有较高亲和力。结论成功构建并表达m97-116-scFv205融合单链抗体,该抗体与iDC具有较高的亲和力,为下一步靶向iDC诱导AChR特异性免疫耐受治疗重症肌无力奠定了良好的实验基础。Objective To construct prokaryotic expression vector of mice acetylcholine receptor peptide（m97-116）and CD205 fusion single chain antibody fragment（m97-116-scFv205）,and to analyze the affinity of the fusion protein to immature dendritic cell（iDC）.Methods Bone marrow cells were harvested from the femur and tibiae of C57BL/6 mice and washed in RPMI 1640 following lysis of red blood cell.Remaining cells were cultured in complete medium（RPMI 1640 supplemented with 5% FCS,）with 10ng/mL GM-CSF and 10ng/mL IL-4.The cells were stimulated with LPS（1ng/mL）or without LPS for 24 hours.The morphology of the cells was observed by the microscope and scanning electron microscopy.Flow cytometry（FCM）was used to analyze the expression of lineage and specific marker.The fragment m97-116-scFv205 was cloned into prokaryotic expression vector pET22b（＋）and the protein expression was induced by 0.1mmol/L IPTG at 18℃for 18 h,and then purified by Ni column via His Tag.Protein production and purification steps were analyzed by SDSPAGE and Western blot.The affinity of m97-116-scFv205 with iDC was tested by FCM via FITC-anti His Tag.Results The morphological and phenotypical analysis showed that cultured cells were typical iDC.The expression of m97-116-scFv205 was demonstrated by SDS-PAGE and Western blot.The m97-116-scFv205 showed high affinity with iDC.Conclusions The prokaryotic expression vector of m97-116-scFv205 was successfully constructed and the m97-116-scFv205 can specifically bind to iDC,providing basis for further studies inducing AChR specific immunologic tolerance through targeted iDC.

关 键 词：重症肌无力 未成熟树突状细胞 融合单链抗体 CD205 

分 类 号：R746.1[医药卫生—神经病学与精神病学]