大肠癌噬菌体抗体库的构建及抗CEA抗体的筛选

Construction of Phage Display Antibody Library of Colorectal Cancer and Screening CEA Phage Antibody

目的构建及初步鉴定大肠癌噬菌体抗体Fab呈现库,筛选人CEA单克隆抗体,并进行序列分析。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PCR扩增人全套抗体基因片段,克隆于pComb3载体,再经电穿孔转化大肠杆菌XL1-Blue菌株,形成噬菌体抗体库。以固相化CEA抗原淘筛抗体库,ELISA鉴定噬菌体抗体。其中一个阳性克隆进行测序。结果逆转录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,Fab基因重组率为50%。以单抗捕获的CEA抗原淘洗4轮,出现特异性富集,阳性克隆经直接ELISA和交叉反应ELISA实验证实具有良好的抗CEA抗原特异性。DNA测序表明该抗体重链属IgG亚类并含有一条IgL亚类的轻链。结论成功构建了大肠癌患者自然致敏抗体Fab段噬菌体呈现库,从中获得可与CEA抗原结合的噬菌体抗体,由此为大肠癌早期诊断及基因治疗提供了一种新的思路和方法。

Objective To construct the Fab phage display antibody library of colorectal cancer so as to screen carcinoembryonic antigen （CEA） phage antibody from the constructed phage display library. Methods The total cell RNA was extracted from a colorectal cancer patient＇s peripheral lymphocytes, and reverse-transcribed into cDNA. These cDNA were amplified to the light and the heavy chain DNA by PCR using relevant primers, and then cloned into the expression pComb3 vector. Through transformed into XL1-Blue Escherichia coli by electroporation, the phage display antibody library was constructed successively. After that, CEA antigen was coated to pan the constructed library in order to select antigen binders, and the selected phage antibodies were assayed by ELISA analysis. One positive clone was analyzed by DNA sequenced. Results The amplified fragments of κ ,λ and Fd DNA were about 680bp. The amplification products were cloned into the expression pComb3 vector and were expressed in XL1-Blue Escherichia coli. A human Fab antibody library was constructed with a size of 2.1× 10^7 and an efficacy of about 50% （ κ ,λ and Fd DNA were all inserted）. Using coated CEA antigen to pan the phage display antibody library four＇rounds, the phage antibodies showed enrichment specifically. The positive clones had good anti-CEA specificity which verified by ELISA directly and cross-reaction. Through DNA sequencing of one positive clone, it showed that its heavy chain belonged to IgG subvariety and its light chain to IgL subvariety. Conclusion We succeed in constructing a Fab phage display antibodies library with natural immunity by a colorectal cancer patient. From it, we also succeed in obtaining the antibody which has the binding activity to CEA antigen.