哺乳动物细胞表面展示全长类风湿关节炎抗体库的构建  

Construction of rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries

作　　者：周烨[1] 陈振瑞[2] 贺微 娄海波[2] 张哲欢[2] 刘叔文 姜世勃 吴曙光 李长征[5] 周辰 

机构地区：[1]南方医科大学基因工程研究所,广东广州510515 [2]南方医院,广东广州510515 [3]药学院,广东广州510515 [4]中医药学院,广东广州510515 [5]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 [6]香港Dgen生物技术有限公司,香港999077

出　　处：《南方医科大学学报》2011年第8期1369-1373,共5页Journal of Southern Medical University

基　　金：香港Dgen Biotech Ltd科研基金;广东省医学科研基金(A2011363)

摘　　要：目的采用哺乳动物细胞表面展示技术构建全长人源类风湿关节炎抗体库。方法分离类风湿关节炎患者的外周血淋巴细胞,提取总RNA,采用RT-PCR的方法扩增抗体全长轻链基因和重链可变区基因,分别插入哺乳动物细胞表达载体pDGB-HC-TM,电击转化感受态大肠杆菌TOPO-10,分别构建抗体轻链基因库和抗体重链基因库,然后将抗体轻链、重链基因库联合转染293T细胞,用流式细胞仪分析全长人源抗体在293T细胞表面的表达。结果成功构建了类风湿关节炎患者来源的IgG1-Kappa型抗体基因库,随机挑选单克隆经DNA序列分析显示轻链库、重链库的序列正确率分别为80%和60%,可表达的抗体库多样性为6.13×1010。结论类风湿关节炎抗体基因库转染293T细胞后能够在细胞表面表达全长人源抗体,所展示的抗体库具有良好的多样性,能够为下一步筛选特异性抗体奠定基础。Objective To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.Methods Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis.The repertoires of kappa light chain（LCκ） and heavy chain variable region（VH） of the antibodies were amplified by RT-PCR.The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately,and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries.293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry.Results The libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain（IgG1） were constructed.The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry.With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%,respectively,as shown by DNA sequence analysis of the randomly selected clones,the libraries showed an expressible combinatory diversity of 6.13×1010.Conclusion The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.

关 键 词：哺乳动物细胞表面展示 类风湿关节炎 抗体基因库 全长人源抗体 流式细胞仪 

分 类 号：R593.22[医药卫生—内科学]