| 全人源抗乳腺癌噬菌体单链抗体库的构建 |
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| 引用本文： | 王净,王慧,袁媛,李青. 全人源抗乳腺癌噬菌体单链抗体库的构建[J]. 现代肿瘤医学, 2011, 19(11): 2139-2142. DOI: 10.3969/j.issn.1672-4992.2011.11.01 |
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| 作者姓名： | 王净 王慧 袁媛 李青 |
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| 作者单位： | 第四军医大学病理与病理生理学教研室,陕西,西安,710032 |
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| 基金项目： | 国家自然科学基金资助项目，国家"重大新药创制"科技重大专项资助项目 |
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| 摘 要： | 目的:利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法:从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞(PBMC),提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链(VH)和轻链(VL)基因,经重叠延伸PCR(SOE-PCR),在体外将两者连接成单链抗体(scFv)基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果:得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论:本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。
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| 关 键 词： | 全人源抗体库 乳腺癌 单链抗体(scFv) 噬菌体抗体库 |
Construction of human phage display scFv library against mastocarcinoma |
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| WANG Jing,WANG Hui,YUAN Yuan,LI Qing. Construction of human phage display scFv library against mastocarcinoma[J]. Journal of Modern Oncology, 2011, 19(11): 2139-2142. DOI: 10.3969/j.issn.1672-4992.2011.11.01 |
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| Authors: | WANG Jing WANG Hui YUAN Yuan LI Qing |
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| Affiliation: | WANG Jing,WANG Hui,YUAN Yuan,LI Qing Department of Pathology and Physiopathology,the Fourth Military Medical University,Shaanxi Xi'an 710032,China |
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| Abstract: | Objective:To construct a human anti-mastocarcinoma cells phage single-chain Fv antibody library by phage display technology.Methods:About 30 peripheral blood samples of breast cancer patients without chemotherapy were got from hospital.Then RNA was isolated from the peripheral blood mononuclear cells(PBMC)and reverse transcribed to cDNA by RT-PCR.VH and VL were amplified and linked together with a linker segment by overlapping PCR(SOE-PCR)to form a human scFv gene.Cut the scFv gene and phagmid vector pCanta... |
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| Keywords: | human antibody library breast cancer scFv phage display antibody library |
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