鼠源抗haPrP23—231噬菌体抗体库的构建和初步鉴定  

Generation and Identification of Phage Engineering Antibodies Library against Hamster Prion Protein

作　　者：黄银霞[1] 韩俊[1] 董辰方[1] 孙力[1] 高晨[1] 王小凡[1] 韩露[1] 周伟[1] 张宝云[1] 姜慧英[1] 梁米芳[1] 董小平[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052

出　　处：《中华实验和临床病毒学杂志》2007年第3期211-213,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金委资助项目（30500018、30571672）;欧盟资助项目（QLRT200001441）和国家科技攻关计划资助项目（2003BA712A04.02）

摘　　要：目的构建和鉴定鼠源抗haPrP23—231噬菌体抗体库。方法利用纯化的朊蛋白抗原免疫BALB／c小鼠，取脾，提取细胞总RNA，逆转录cDNA，以其为模板进行免疫球蛋白Fab区基因的特异性PCR扩增，将轻链和重链Fd段基因分别构建到pComb3质粒，构建噬菌体抗体库。结果经过四轮“吸附-洗脱-富集”的过程后获得了12株阳性克隆。挑取5株进行序列分析，并将结果与GenBank中已知序列库中的IgG序列进行比较，得到了两株不同的抗体，并进行了初步鉴定。结论本研究为朊病毒病的研究奠定了基础。Objective Generation and Identification of Phage Engineering Antibodies Library against Hamster Prion Protein. Methods Fab antibodies were identified and confirmed. BALBIc mice were immuned with PrP proteins. After the third immunization, the total RNA was extracted from the mice spleens. The genes of heavy Fd Fragmenst and light chain of antibodies amplified by a series of specific primers of human IgG Fab fragment were cloned into phagmid vesctor pComb3. Results The combinatorial Fab library were constructed successfully and the cloning efficiencies both of light chain and Fd fragments were about near 10^6 . The Fab library were panned by four cycles and screened with purified haPrP23-231 antigen on microtiter plates. 12 mAbs were isolated after four cycles of panning, five of which were sequenced and resulted sequence data were analyzed by alignment with GenBank immunoglobulin genes. Two strain of new heavy and light chain genes of Fab antibodies were identified and confirmed. Conclusion The research in this article will provide foundation for study of diagnosis and therapy of prion.

关 键 词：朊病毒 肽库 遗传工程 细菌噬菌体 

分 类 号：R3[医药卫生—基础医学]