人AChR-Fc融合蛋白真核表达载体的构建和表达  

Construction and expression of a fusion protein containing extracellular domain of human AChRα1 and Fc fragment of human IgG1

作　　者：常婷[1] 林宏[1] 刘煜[1] 李柱一[1] 

机构地区：[1]第四军医大学唐都医院神经内科,710038

出　　处：《中国神经免疫学和神经病学杂志》2011年第4期276-279,共4页Chinese Journal of Neuroimmunology and Neurology

基　　金：国家自然科学基金面上资助项目(30870841)

摘　　要：目的构建含人AChRα1亚单位胞外段(Hα1-210)-IgG1Fc融合基因的真核表达载体,表达人AChR-Fc融合蛋白。方法扩增Hα1-210基因,插入含有CMV启动子、小鼠Kappa链先导序列以及人IgG1从铰链区到CH3基因片段的真核表达载体pAN1782中,构建重组表达载体pAN-Hα1-210,并经酶切、测序鉴定;采用脂质体法将重组载体转染CHO-K1细胞,经G418加压筛选、ELISA检测,挑选高表达融合蛋白的阳性转化子扩大培养;表达的AChR-Fc融合蛋白经Protein A亲和层析柱纯化,SDS-PAGE、Western blot鉴定。结果酶切及测序结果证实Hα1-210基因序列正确,真核表达载体pAN-Hα1-210构建成功;ELISA检测证实AChR-Fc融合蛋白在细胞培养上清中呈分泌表达;SDS-PAGE结果显示该融合蛋白的相对分子质量约为51 000;Western blot结果显示该融合蛋白能被特异性单克隆抗体mAb198所识别。结论成功构建pAN-Hα1-210真核表达载体,并获得具有生物活性的AChR-Fc融合蛋白,为进一步开展融合蛋白靶向B细胞治疗重症肌无力的研究奠定了基础。Objective To construct eukaryotic expression vector containing the extracellular domain of the human AChRal-subunit （Hod-210） and Fc fragment of human IgG1, and to express human AChR-Fc fusion protein. Methods The extraeellular domain of Hal-210 was amplified and inserted into pAN1782 eukaryotic expression vector with a cytomegalovirus promoter, a murine imrnunoglobulin k-chain leader sequence end the human genomic IgG 71 constant region from the hinge to the end of CH3. Recombinant expression vector pAN- Hal-210 was constructed and evaluated by restriction enzyme analysis and sequencing. The right pAN-Hal-210 plasmid was transfected into CHO-K1 cells with lipofectin reagent and selected by G418. The positive clone with high expressing fusion protein were selected through ELISA detection and cultured. After purified by protein A affinity column chromatography, the AChR-Fe fusion protein was identified by SDS-PAGE and Western blot assay. Results Restriction enzyme and sequencing indicated Hal-210 gene sequence was correct and pAN-Hal- 210 had been constructed succeSsfully. AChR-Fe fusion protein could be detected by ELISA assay in the CHO-K1 culture supernatant. There was only one special band at the position of relative molecular mass 51 000 by SDS- PAGE assay, and it was equivalent to expected value. Western blot analysis also showed that fusion protein eould react to mAb198. Conclusions We successfully constructed eukaryotie expression vector pAN-Hal-210 and obtained AChR-Fe fusion protein with biological activity, thus lay a foundation for further works on the treatment of myasthenia gravis （MG） by targeting B cells.

关 键 词：重症肌无力 融合蛋白 真核表达 CHO—K1细胞 

分 类 号：R746.1[医药卫生—神经病学与精神病学]