抗血管内皮生长因子受体2双价单链抗体的构建表达及其活性研究

中国生物工程杂志

2011, Vol. 31

Issue (8): 1-6

研究报告

抗血管内皮生长因子受体2双价单链抗体的构建表达及其活性研究

丁笠, 王秀云, 齐海迪, 李海鑫, 周雅琼, 陈耀祖, 张娟, 王旻

中国药科大学生命科学与技术学院南京 210009

Construction,Expression and Biological Activity Studies of a Bivalent Single Chain Variable Fragment(BsFv)against Vascular Endothelial Growth Factor Receptor 2

DING Li, WANG Xiu-yun, QI Hai-di, LI Hai-xin, ZHOU Ya-qiong, CHEN Yao-zu, ZHANG Juan, WANG Min

School of Life Science & Technology,China Pharmaceutical University,Nanjing 210009,China

全文: PDF(835 KB) HTML

摘要：

为了竞争性抑制血管内皮生长因子(VEGF)与其受体(VEGFR-2)的结合,构建表达了能够与VEGFR-2胞外3区(KDR3)特异结合的双价单链抗体(bivalent single-chain Fv,BsFv),并鉴定其生物活性。以KDR3特异结合单链抗体AK404(scFv AK404)基因为模板,设计引物引入中间连接肽(G₄S)₃连接两个scFv片段,将其克隆入原核表达载体pET22b,并转化至大肠杆菌BL21(DE3)中诱导表达,表达产物依次经过镍柱亲和层析纯化和透析复性;基于生物膜层表面干涉技术(Bio-Layer Interferometry,BLI)技术的体外亲和力监测实验和VEGF诱导的人脐静脉内皮细胞(HUVEC)增殖实验鉴定表达产物与KDR结合的活性。酶切鉴定和DNA测序结果表明BsFv构建成功;SDS-PAGE显示37 ℃下1 mmol/L IPTG诱导细菌6 h获得包涵体,经镍柱亲和层析纯化和透析复性得到纯度为90%的BsFv;经Western blotting鉴定为目的蛋白;体外结合实验表明:BsFv特异性结合KDR胞外3区的亲合力约为单价抗体AK404的2倍;内皮细胞增殖抑制实验表明:BsFv可剂量依赖性地抑制HUVEC细胞增殖,且其抑制效果强于AK404。上述结果表明:BsFv在抗血管生成活性方面比其scFv具有明显优势,为进一步用于抗血管生成抗肿瘤治疗和肿瘤诊断奠定了基础。

关键词： 血管内皮生长因子受体2; 双价单链抗体; 生物膜层表面干涉技术; 抗血管生成抗肿瘤治疗

Abstract:

It was focused on that,the binding efficiency of Vascular Endothelial Growth Factor(VEGF)and Vascular Endothelial Growth Factor Receptor-2(VEGFR-2)competitively inhibited by bivalent single-chain Fv(BsFv),and BsFv's construction,expression and bioactivity identification. PCR primers were designed to introduce interlinker(G₄S)₃ between two scFv AK404 fragments,which were ligated with vector pET22b and transformed into E.coli BL21(DE3).The expression product was purified by IMAC and renatured by dialysis. The KDR-binding activity was determined by Bio-Layer Interferometry(BLI)and its influence on the propagation of HUVEC cell induced by VEGF. Bsfv was successfully constructed,and expressed as inclusion body after having been induced by 1 mmol/L IPTG for 6h at 37 ℃. After purification by IMAC and renaturation of the denatured recombinant protein,the BsFv with bioactivity and 90% purity was obtained. It was characterized to be target protein by Western blotting. The result of binding experiment in vitro based on BLI had proved that BsFv has higher binding ability to KDR3 than AK404. Additionally,BsFv was shown to inhibit the HUVEC cell's proliferation in a concentration-dependent manner,and the inhibition rate was relatively higher than that of AK404. All the results indicated that BsFv have higher anti-KDR activity compared with scFv,which provided a potential candidate drug in further research on its application on antiangiogenic oncotherapy and tumor diagnostics.

Key words: Vascular endothelial growth factor receptor 2 Bivalent single chain Fv Bio-layer interferometry Antiangiogenic oncotherapy

收稿日期: 2011-04-13 出版日期: 2011-08-25

ZTFLH:

Q789

基金资助:

国家自然科学基金(81072561)、江苏省自然科学基金(BK2009299)、江苏省普通高校研究生科研创新计划(CX10B-376Z)资助项目

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引用本文:

丁笠, 王秀云, 齐海迪, 李海鑫, 周雅琼, 陈耀祖, 张娟, 王旻. 抗血管内皮生长因子受体2双价单链抗体的构建表达及其活性研究[J]. 中国生物工程杂志, 2011, 31(8): 1-6.

DING Li, WANG Xiu-yun, QI Hai-di, LI Hai-xin, ZHOU Ya-qiong, CHEN Yao-zu, ZHANG Juan, WANG Min. Construction,Expression and Biological Activity Studies of a Bivalent Single Chain Variable Fragment(BsFv)against Vascular Endothelial Growth Factor Receptor 2. China Biotechnology, 2011, 31(8): 1-6.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I8/1

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