大肠癌噬菌体抗体库的构建及初步鉴定  

Construction and identification of the phage display antibody library of human colorectal cancer

作　　者：南清振[1] 高蕾 肖冰[1] 杨希山[1] 张振书[1] 

机构地区：[1]南方医科大学南方医院消化内科,510515 [2]广州中医药大学第二附属医院影像科,510120

出　　处：《现代消化及介入诊疗》2008年第1期1-5,共5页Modern Interventional Diagnosis and Treatment in Gastroenterology

摘　　要：目的构建及初步鉴定大肠癌噬菌体抗体Fab呈现库。方法分离大肠癌患者外周血淋巴细胞,提取淋巴细胞总RNA,逆转录成cDNA。用相应引物进行PCR,扩增出轻链和重链Fd段基因,经酶切后分别和噬粒载体pComb3连接,再经电穿孔转化大肠杆菌XL1-Blue菌株,将轻链和重链Fd基因先后克隆入pComb3中。结果从分离出的淋巴细胞中提取到高质量RNA,RT-PCR分别扩增出约680bp大小的κ、λ和Fd基因。PCR产物和载体经纯化、双酶切后进行连接转化,成功地构建了人源性Fab抗体基因库,库容量达2.1×107,轻链κ、λ和重链Fd基因均插入pComb3的重组率为50%。结论构建了大肠癌患者自然致敏抗体Fab段噬菌体呈现库,为筛选大肠癌相关抗体打下基础。Objective To construct and to identify the Fab phage display antibody library of the colorectal cancer. Methods Total cell RNA extracted from peripheral lymphocytes of a colorectal cancer patient was reverse-transcribed into cDNA. The light chain gene and the heavy chain Fd gene were amplified by PCR using relevant primers, the amplification products and the expression vector pComb3 were ligased with T4 DNA ligase after had been cut with an excess of the restriction enzymes. Following ligation, they were transformed by electroporation into Escherichia coli XL1-Blue, and the light chain genes and the heavy chain Fd gene were inserted successively into the expression vector pComb3, respectively. Results The amplified fragments of K, k and Fd genes by RT-PCR were about 680 bp. The amplification products and the expression vector pComb3 were ligased and were transformed by electroporation after purification, and were cut with the restriction en- zymes. A human library of Fab antibodies was constructed, with the size being 2.1 × 10^7. Recombination ratio of 50% was got, and the K, k and Fd genes were all inserted. Conclusion A natural immune Fab antibody phage display library of human colorectal cancer was constructed. Which may be useful for screening the related antibodies for colorectal cancer.

关 键 词：大肠癌 噬菌体抗体库 FAB抗体 

分 类 号：R735.34[医药卫生—肿瘤] R392.11[医药卫生—临床医学]