SARS冠状病毒N蛋白在酵母中的表达纯化及抗原性分析  

Expression,purification and antigen analysis of SARS-associated coronavirus nucleocapsid protein in Pichia pastoris system

作　　者：郭旭[1] 张竞[2] 李玉珍[1] 张麒[3] 李东云[3] 

机构地区：[1]解放军总医院麻醉科,北京100853 [2]解放军总医院胸外科,北京100853 [3]解放军总医院呼吸科,北京100853

出　　处：《第三军医大学学报》2006年第1期59-62,共4页Journal of Third Military Medical University

摘　　要：目的研究人SARS冠状病毒核壳N蛋白在Pichia pastoris酵母中的表达,获得具有生物学活性的重组N蛋白,并鉴定表达蛋白的抗原性,为下一步的研究奠定基础。方法合成引物扩增SARS冠状病毒N基因,插入含AOX1启动子的Pichia pastoris酵母表达载体中,转化酵母宿主菌KM71H、X-33、GS115,筛选阳性转化子,摇瓶培养,用甲醇诱导表达。表达产物经镍离子柱亲和层析纯化后,以抗SARS冠状病毒N蛋白的小鼠单抗、兔多抗及SRAS病人阳性血清鉴定其抗原性。结果SDS-PAGE和免疫学分析显示,酵母转化子仅在KM71H、X-33中有M r约70 000的诱导蛋白,表达产物具有良好的抗原性和特异性。结论在酵母表达系统中,初步实现了SARS冠状病毒核壳N蛋白的有效表达。Objective To study the expression of SARS-associated coronavirus nucleocapsid （N） gene in Pichia pastoris and to obtain recombinant N protein with good biological activity. Methods The gene encoding the full N protein of SARS-CoV was amplified by PCR and cloned into Pichia pastoris expression vector pPICZA. The recombinant expression plasmid pPICZA/N was transformed into X-33, KM71H and GS115. The positive insert transformants were screened, cultured and induced by methanol. The recombinant protein was further purified with Ni affinity chromatography. Antigen activity was detected with anti-N monoclonal antibody, polyclonal antibody and sera from SRAS patients. Results SDS-PAGE and immunological analysis demonstrated that only Pichia pastoris transformants KM71H/pPICZA/N and X-33/pPICZA/N expressed Mr 70 000 fusion protein with special antigenicity. Conclusion SARS-CoV N protein expression in Pichia pastoris has been achieved, establishing the basis for further study of biological and immunological function of N antigen.

关 键 词：SARS冠状病毒 N蛋白 基因表达 毕赤巴斯德酵母 

分 类 号：R373.9[医药卫生—病原生物学] R392.11[医药卫生—基础医学]