采用哺乳动物细胞表面展示技术构建全长人源抗肾癌抗体基因库  

Construction of human full-length renal cell carcinoma patient-specific antibody library by mammalian cell surface display

作　　者：陈振瑞[1] 李长征[2] 贺微[3] 周烨[2] 张哲欢[1] 刘叔文[3] 谭万龙[1] 周辰 

机构地区：[1]南方医科大学南方医院泌尿外科,广东广州510515 [2]南方医科大学基因工程研究所,广东广州510515 [3]南方医科大学药学院抗病毒中心,广东广州510515 [4]香港Dgen生物技术有限公司,香港999077

出　　处：《南方医科大学学报》2010年第5期1059-1062,1065,共5页Journal of Southern Medical University

基　　金：香港Dgen Biotech Ltd科研基金

摘　　要：目的采用哺乳动物细胞表面展示技术构建全长人源抗肾癌抗体基因库。方法分离肾癌患者的外周血淋巴细胞(PBMC),提取PBMC的总RNA,采用RT-PCR的方法扩增抗体全长Kappa型轻链(LCκ)和重链可变区(VH)基因,分别插入载体pDGB-HC-TM,电击转化感受态大肠杆菌TOPO10,构建轻、重链抗体基因库,然后将轻、重链抗体基因库联合转染293T细胞,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IgG1-Kappa型抗肾癌抗体基因库,随机挑选的克隆经DNA序列分析显示轻、重链库的序列正确性达90%(9/10)和80%(8/10),流式细胞仪分析显示来自轻、重链库的上述克隆各有7个可检测到相应基因在293T细胞表面的表达,可表达抗体库库容量高达7.5×1010。结论 IgG1-Kappa型抗肾癌抗体基因库转染293T细胞后能够在细胞表面表达全长人源抗体,抗体的多样性为下一步筛选特异性抗体奠定良好的基础。Objective To construct human renal cell carcinoma patient-specific full-length antibody library using mammalian cell surface display technique. Methods Peripheral blood mononuclear cells （PBMC） were isolated from patients with renal cell carcinoma. The repertoires of kappa light chain （LCκ） and heavy chain variable region （VH） of antibody were amplified by RT-PCR. The LCκ and VH libraries were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO10 to construct the renal cell carcinoma patient-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length human antibodies expressed on the surface of 293T cells were analyzed by flow cytometry. Results The libraries of renal cell carcinoma-specific antibody kappa light chain （LCκ） and heavy chain （IgG1） were constructed. The expression of the full-length human antibodies on the surface of 293T cell was confirmed by flow cytometry. The libraries showed an expressible combinatory diversity of 7.5 ×1010. Conclusion The expressible antibody library provides a useful platform for screening of renal cell carcinoma-specific antibodies.

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

分 类 号：R737.11[医药卫生—肿瘤]