重组表达人乙酰胆碱受体α1亚基胞外域蛋白  

Expression and Refolding of The Extracellular Domain of The Human Acetylcholine Receptor α1 Subunit

作　　者：孙辰婧[1] 徐江[1] 李柱一[1] 

机构地区：[1]第四军医大学唐都医院神经内科,陕西西安710038

出　　处：《中国神经免疫学和神经病学杂志》2009年第1期35-38,共4页Chinese Journal of Neuroimmunology and Neurology

基　　金：国家自然科学基金资助项目(30570641)

摘　　要：目的获得大量正确折叠的重组人乙酰胆碱受体(AChR)α1亚基N末端胞外域(ECD)蛋白。方法用RT-PCR方法从TE671细胞中扩增出AChRα1亚基全序列,在此基础上再扩增出ECD基因序列并将其插入到原核表达载体pET16b。以构建的新载体转化E.coliBL21(DE3)pLysS,培养物以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并通过SDS-PAGE比较诱导前后蛋白表达情况。重组蛋白以Ni2+亲和层析纯化。蛋白透析复性后,以ELISA法检测活性。结果PCR产物大小为650 bp,与预计相符;所构建质粒经测序证实ECD核苷酸序列正确,并正确插入载体pET16b。诱导所产生的大量包涵体蛋白经复性后正确折叠且具活性。结论可通过原核表达获得大量正确折叠的可溶性重组人肌肉AChRα1亚基ECD蛋白。Objective To obtain large quantities of correctly folded recombinant extracellular domain （ECD） of human skeletal muscle acetyleholine receptor （AChR） α1 subunit. Methods The full length cDNA of human skeletal muscle AChR α1 subunit encompassing the coding sequence （CDS） was amplified by RT-PCR from TE671 cells. The DNA fragment encoding ECD was amplified by PCR and inserted into the prokaryotic expression vector pET-16b. The reconstructed plasmid was transformed into the host strain BL21 （DE3） pLysS. Protein expression was induced by isopropy-β-D-tbiogalactoside （IPTG） and analyzed by SDS-PAGE. The expressed protein was purified by immobilized Ni^2＋ affinity chromatography and refolded by dialysis. The refolded protein was identified by ELISA. Results A 650 bp band was amplified which was as expected. Sequencing results of the reconstructed plasmid showed no mutation or frame shift. Inclusion bodies were resolved and refolded. The identity of the refolded protein was confirmed by ELISA probed with a specific antibody mAb35. Conclusions It is feasible to obtain large quantities of correctly folded recombinant ECD of human muscle AChR.

关 键 词：重症肌无力 乙酰胆碱受体 N-末端胞外域 主要免疫原区 mAb35 

分 类 号：R746.1[医药卫生—神经病学与精神病学]