AChR-IgG Fc段融合蛋白真核表达载体的构建及表达  

Construction of eukaryotic expression vector of AChR-IgG Fc fragment fusion protein and its expression in CHOk1 cells

作　　者：高洁[1] 常婷[1] 王键[2] 李柱一[1] 

机构地区：[1]第四军医大学唐都医院神经内科,710038 [2]第四军医大学全军神经科学研究所,710038

出　　处：《中国神经免疫学和神经病学杂志》2010年第3期188-191,共4页Chinese Journal of Neuroimmunology and Neurology

基　　金：国家自然科学基金资助项目(30870841)

摘　　要：目的构建人AChR-IgG Fc段融合蛋白真核表达载体AChR-IgG Fc/pAN1782并在CHOk1细胞中表达AChR-IgG Fc融合蛋白。方法以人烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor,nAChR)α1亚基全序列为模板,以PCR法扩增AChRα1亚基胞外段主要免疫区基因片段Hα1-121,将其插入真核表达载体pAN1782。将构建的新载体转染至CHOk1细胞,建立稳定表达AChR-IgG Fc融合蛋白的CHOk1细胞株,以Western blot法检测AChR-IgG Fc融合蛋白表达。结果 (1)Hα1-121经PCR法扩增后所获428 bp基因片段大小符合预计结果,测序所得核苷酸序列与人类基因库中AChRα1亚基胞外段基因片段Hα1-121序列完全一致,未出现点突变或移码突变。所构建的新载体经酶切鉴定证实片段插入正确,载体构建成功。(2)Westernblot法检测结果显示转染新载体的CHOk1细胞培养上清液中有AChR-IgG Fc融合蛋白表达。结论成功构建人AChR-IgG Fc段融合蛋白真核表达载体,且该融合蛋白可在转染新载体的CHOk1细胞中稳定表达,为下一步靶向B细胞治疗重症肌无力奠定了初步良好基础。Objective To construct an eukaryotic expression vector of AChR-IgG Fc, and to express the fusion protein in CHOkl cells. Methods The genomic fragment Ha1-121 of the human skeletal muscle acetyleholine receptor （ACbR） a1 subunit was amplified by PCR and inserted into the eukaryotic expression vector pAN1782. The recombinant plasmid was transfected into CHOkl cells with screening culture by G418 for stable expression. The expression of the AChR IgG Fc fusion protein was detected by Western blot. Results A 428 bp band was amplified as expected and the sequencing results showed no mutation or frame shift. The recombinant plasmid was confirmed by enzyme digestion and transfected into CHOkl cells. The stable expression of the AChR-IgG Fc fusion protein was demonstrated by Western blot. Conclusions The eukaryotic expression vector of AChR-IgG Fc/pAN1782 was successfully constructed and the AChR-IgG Fc fusion protein was stably expressed in CHOkl cells, providing basis for the B cell targeted therapy of myasthenia gravis.

关 键 词：重症肌无力 受体 胆碱能 IGG FC段 融合蛋白 

分 类 号：R746.1[医药卫生—神经病学与精神病学]