抗H5亚型禽流感病毒单链抗体在毕赤酵母中的分泌型表达和生物活性分析  

Secretory Expression and Biological Activity Analysis of an Anti-H5 Single-chain Antibody from Pichia Pastoris

作　　者：何芳萍[1] 林庆山[1] 李少伟[1] 魏旻希[1] 陈振钦[1] 罗文新[1] 陈毅歆[1] 张军[1] 夏宁邵[1] 

机构地区：[1]厦门大学国家传染病诊断试剂与疫苗工程技术研究中心,生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室,厦门361005

出　　处：《病毒学报》2011年第3期202-206,共5页Chinese Journal of Virology

基　　金：国家高技术研究发展计划重点项目(863计划)(No.2006AA020905);国家自然科学基金项目(No.30901077,30972826);重大传染病专项及新药创制(No.2008ZX10004-006,2009ZX09102-230,2009ZX10004-704)资助

摘　　要：在本实验室研制出的多株针对H5N1亚型禽流感血凝素单抗中,13D4单抗对所有H5亚型病毒均有血凝抑制和中和活性,具有特异性高、反应性强和识别广的特点,且在小鼠实验中显示了对各种代表株禽流感的感染和发病均具有良好的治疗效果。在此研究基础上,本实验通过基因工程构建含有13D4单链抗体(scFv)基因的毕赤酵母表达载体,实现目的蛋白的分泌性表达和纯化。经过竞争法和血凝抑制检测其活性,表明获得的单链抗体具有与原始鼠源抗体相近的反应活性和相同的识别表位。H5N1广谱中和单抗13D4的单链抗体的成功构建,为进一步研制针对H5N1禽流感病毒的治疗性抗体奠定了基础。In our previous study,a panel of 52 broadly cross-reactive H5-specific monoclonal antibodies（MAbs） were generated and characterized.The 13D4,one of these MAbs,has been demonstrated to protect mice against lethal challenge by 4 strains of H5N1 avian influenza virus representing the currently prevailing genetic populations,clades 1,2.1,2.2,and 2.3.Here,we further cloned the gene of the 13D4 MAb and constructed a single-chain variable fragment.Then,the 13D4 single-chain antibody（scFv） was expressed in secretory maner in Pichia pastoris.The supernatant of the culture was concentrated and subjected to ammonium sulfate precipitation.The purity of the 13D4 scFv was around 90% in SDS-PAGE following ion-exchange chromatography.We further investigated its binding property using hemagglutination inhibition（HI） test and blocking ELISA.The results indicated that the 13D4 scFv shared the same binding sites and comparable HI titer with the prototype murine 13D4 Mab.In conclusion,an anti-H5 single-chain wide-spectrum neutralizing antibody is prepared successfully in yeast system.

关 键 词：高致病性禽流感病毒 单链抗体 血凝抑制 毕赤酵母 

分 类 号：Q786[生物学—分子生物学] R373.1[医药卫生—病原生物学]