哺乳动物细胞表面展示人源HIV特异性全长抗体库的构建  

Construction of HIV-specific Full-length Human Antibody Library

作　　者：周烨[1] 李长征[2] 陈振瑞[3] 贺微[4] 杨于军 刘叔文[4] 吴曙光[4] 马丽英 姜世勃[4,7] 周辰[4,5] 

机构地区：[1]南方医科大学基因工程研究所,广东广州510515 [2]南方医科大学中医药学院,广东广州510515 [3]南方医科大学南方医院,广东广州510515 [4]南方医科大学药学院,广东广州510515 [5]香港Dgen生物技术有限公司,香港999077 [6]中国疾控中心性病艾滋病中心,北京100050 [7]复旦大学上海医学院分子病毒学教育部/卫生部重点实验室,上海200032

出　　处：《中国医药指南》2011年第21期5-7,共3页Guide of China Medicine

基　　金：Dgen Biotech Ltd科研基金(DGB12012008)

摘　　要：目的构建人源的HIV特异性全长抗体基因库并获得可稳定展示全长抗体的哺乳动物细胞库。方法提取HIV患者外周血淋巴细胞的总RNA,采用RT-PCR的方法扩增抗体重链可变区(VH)基因,插入哺乳动物细胞表达载体pDGB4,转化感受态大肠杆菌TOPO-10,构建抗体基因库,随后将抗体库的质粒DNA稳定转染FCHO细胞,获得可稳定展示全长抗体的细胞库,用流式细胞仪分析全长抗体在FCHO细胞表面的表达。结果本研究以少量外周血淋巴细胞RNA为来源,构建了库容量为7.77×104的全长抗体基因库;将基因库的质粒DNA稳定转染FCHO细胞后,获得了可稳定展示全长抗体的细胞库;经流式细胞仪分析,有67%的细胞表面可表达可被检测到的全长抗体。结论本研究构建了人源的HIV特异性全长抗体基因库并获得可稳定展示全长抗体的哺乳动物细胞库,为治疗HIV感染的特异性抗体的筛选打下了基础。Objective To construct an HIV-specific full-length human antibody gene library and establish a mammalian cell library which can stably display full-length antibodies.Methods The total RNA was isolated from peripheral blood lymphocyte of the patient infected with HIV.The heavy chain variable regions（VH）were amplified by RT-PCR and were then inserted into the vector pDGB4.The ligation mixture was transformed into competent E. coli TOPO-10 cells to construct the antibody gene library and the library size was analyzed.The library DNAs were transfected into FCHO cells to construct the cell library which can stably display full-length antibodies.Expression of the full-length human antibodies on the surface of FCHO cells was analyzed by flow cytometry.Results In this work,we constructed a full-length antibody gene library using a small amount of total RNA isolated from peripheral blood lymphocyte of HIV-infected patient as template,and the library size was 7.77×10^4.After a stable transfection of the antibody gene library into FCHO cells,we successfully constructed a cell library which can stably display full-length antibody.About 67%of the transfected cells expressed the full-length antibodies on cell surfaces.Conclusion A human HIV specific full-length antibody gene library and a FCHO cell library which could stably display full- length antibody on cell surfaces were constructed.These libraries can be used for identification of human anti-HIV antibodies as passive immunotherapeutics.

关 键 词：哺乳动物细胞表面展示 全长抗体库 人源抗体 稳定展示 

分 类 号：R512.91[医药卫生—内科学]