抗人大肠癌双价单链抗体基因的构建及表达
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严丹丹, 杨福辉, 方 瑾
双价单链抗体;ND-1;大肠癌;免疫活性严丹丹,杨福辉,方瑾.抗人大肠癌双价单链抗体基因的构建及表达. 世界华人消化杂志2006;14(24)2395-2400,抗人大肠癌双价单链抗体基因
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严丹丹, 杨福辉, 方瑾, 中国医科大学基础医学院细胞生物学教研室, 卫生部细胞生物学重点实验室 辽宁省沈阳市 110001国家自然科学基金资助项目, No. 85-722-18-02, 20375047
通讯作者: 方瑾, 110001, 辽宁省沈阳市和平区北二马路92号, 中国医科大学细胞生物教研室. jfang61@netease.com
电话: 024-23256666-5347
收稿日期: 2006-06-07 接受日期: 2006-06-30
Construction and expression of bivalent single-chain antibody against human colorectal carcinoma
Dan-Dan Yan, Fu-Hui Yang, Jin Fang
Dan-Dan Yan, Fu-Hui Yang, Jin Fang, Department of Cell Biology, College of Basic Medical Sciences, Key Lab of Cell Biology Affiliated to Ministry of Public Health of China, China Medical University, Shenyang 110001, Liaoning Province, China
Supported by National Natural Science Foundation of China, No. 85-722-18-02, 20375047
Correspondence to: Professor Jin Fang, Department of Cell Biology, China Medical University, 92 Beier Road, Heping District, Shenyang 110001, Liaoning Province, China. jfang61@netease.com
Received: 2006-06-07Accepted: 2006-06-30
Abstract
AIM: To construct the fusion gene of recombinant bivalent single-chain antibody ND-1sc(Fv)2 against human colorectal carcinoma and express it in E. coli.
METHODS: The two same ND-1scFv genes against human colorectal carcinoma were covalently linked by a 15-bp linker with sequences encoding GGGGS. The expression vector pET-28a(+)ND-1sc(Fv)2 was constructed to express the ND-1sc(Fv)2 fusion protein in E. coli. The expressed product was purified by metal affinity chromatography using Ni-NTA resin and then detected by SDS-PAGE and Western blotting. Its immunity was analyzed by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA).
RESULTS: Sequencing results showed that the ND-1sc(Fv)2 gene was 1473 bp in length, including two scFv of 729 bp and one GGGGS of 15 bp. SDS-PAGE and Western blotting showed that the relative molecular mass of fusion protein was about 55 kDa, in accordance with its predicted Mr value ......
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