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浙江大学学报（医学版）
浙江大学学报（医学版）  2014, Vol. 43 Issue (4): 434-440    DOI: 10.3785/j.issn.1008-9292.2014.07.006
专题报道     
人源性抗乳腺癌HER-2噬菌体单链抗体库的构建与筛选
卢晓辉1，王志文1，3，蔡颖1，黄静1，朱丽华1，杨清玲2，陈昌杰2
1. 蚌埠医学院临床检验诊断学实验中心，安徽 蚌埠233000 2. 蚌埠医学院生物化学与分子生物学教研室，安徽 蚌埠233000 3. 河南大学淮河医院检验科，河南 开封 475000
Construction and identification of anti-HER2 phage display single chain fragment of variable region library in human breast cancer
LU Xiao-hui1， WANG Zhi-wen1，3， CAI Ying1， HUANG Jing1， ZHU Li-hua1， YANG Qing-ling2， CHEN Chang-jie2
1. Clinical Testing and Diagnose Experimental Center， Bengbu Medical College， Anhui 233030， China; 2. Department of Biochemistry & Molecular Biology， Bengbu Medical College， Anhui 233000， China; 3. Department of Clinical Laboratory， Huaihe Hospital of Henan University， Kaifeng 475000， China
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摘要: 目的：构建人源性抗乳腺癌噬菌体单链抗体（scFv）库，筛选和鉴定抗乳腺癌人类表皮生长因子受体2（HER2）特异性scFv，为HER2和CXC趋化因子受体4（CXCR4）双靶向融合蛋白的研制提供靶向HER2的高亲和力序列。 方法：取已确诊的乳腺癌患者癌旁淋巴组织，提取总RNA；构建可变区基因的T载体库，将重链可变区（VH）和轻链可变区（VL）基因与pCANTAB连接肽连接，获得pCANTAB-scFv，电转化感受态大肠杆菌TG1；以M13K07辅助噬菌体进行感染，获噬菌体抗体库；利用酶联免疫吸附试验（ELISA）检测筛选后获得的噬菌体抗体活性，以免疫组织化学法检测抗体亲和力，并通过测序进一步鉴定。 结果：成功构建大容量pCANTAB-scFv抗体库，获得的scFv长度为750 bp，VH和VL长度分别为375和330 kb；电转化大肠杆菌TG1后酶切验证插入片段大小正确，得到库容为2.48×108的大容量抗体库；通过噬菌体展示和淘洗筛选，富集针对乳腺癌细胞株SKBR-3表面抗原的抗体库；所得抗体对HER2有高度的亲和力和特异性，对乳腺癌组织HER2抗原也具有较高的亲和力；测序结果与人IgG抗体进行对比，所获得的scFv具有高度的人源性。 结论：主要构建了大容量人源性抗乳腺癌细胞的噬菌体scFv库，并筛选获得可特异性识别人乳腺癌HER2的高亲和力scFv，为制备以HER2为靶点的抗肿瘤HER2和CXCR4双靶向融合蛋白提供了载体。
Abstract：Objective： To construct human phage single-chain antibody （scFv）library against breast cancer， and to identify anti-HER2 specific antibodies from the human phage display scFv library to offer a stronger affinity sequence targeting HER2 for fusion protein targeting HER2 and CXCR4. Methods： Total RNA was extracted from the adjacent lymphatic tissue harvested from breast cancer patients. The variable regions of the whole antibody were amplified by using RT-PCR and were cloned into the vector pCANTAB-5E through a linker. The products were electroporated into competent E.coli TG1 cells. Recombinant phages specific for breast cancer cells were enriched in SKBR-3 after four rounds. The antigen-positive clones were selected by ELISA and immunohistochemistry. Results： The fragment of VH and VL were about 375 and 330 bp and were linked in vitro to form scFv of 750 bp that was resistant to the breast cancer HER2 single strand. A fusion phage display library that contained total of 2.48×108 pfu /ml was established. ELISA and immunohistochemical results confirmed that the antibody has a strong affinity with HER2 antigen in breast cancer tissue. Compared to human IgG antibody， a scFv phage library against human breast cancer was successfully constructed with high capacity. The scFv was highly specific to HER2 antigen and the sequencing results indicated that VL and VH genes were highly homologous with the variable region of human antibody. Conclusion： This strategy may achieve new targeted antibody resistant to the breast cancer for clinical treatment and provide a carrier that uses HER2 as a target of the fusion protein for anti-tumor therapy．
收稿日期: 2014-01-13
基金资助:国家自然科学基金（81071848）；安徽省自然科学基金（1208085MH166）；安徽省教育厅自然科学重点研究项目（KJ2013A184，KJ2010A240）.
通讯作者: 陈昌杰，男，博士，教授，硕士生导师，主要从事肿瘤分子生物学研究；E-mail：tochenchangjie@163.com. 杨清玲，女，硕士，教授，硕士生导师，主要从事肿瘤分子生物学研究；E-mail: yqlmimi@163.com   
Corresponding author: CHEN Chang-jie，E-mail：tochenchangjie@163.com；YANG Qing-ling，E-mail: yqlmimi@163.com   
作者简介: 卢晓辉（1988-），男，硕士研究生，主要从事肿瘤分子生物学研究；E-mail：872422417@qq.com
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引用本文:

卢晓辉，等. 人源性抗乳腺癌HER-2噬菌体单链抗体库的构建与筛选[J]. 浙江大学学报（医学版）, 2014, 43(4): 434-440.
LU Xiao-hui，et al. Construction and identification of anti-HER2 phage display single chain fragment of variable region library in human breast cancer. Journal of ZheJiang University(Medical Science), 2014, 43(4): 434-440.

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http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2014.07.006      或      http://www.zjujournals.com/xueshu/med/CN/Y2014/V43/I4/434

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