人源抗Met基因工程抗体scFv的改造与特性分析
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江苏省社会发展项目资助(BS2007019);南京军区“122”工程资助


Reconstitution of human anti-Met genetic engineering antibody scFv
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    摘要:

    目的:将已制备的抗Met 单链抗体基因与人IgG Fc基因片段融合,表达有活性的融合蛋白分子,以增强单链抗体的可溶性-方法:从人淋巴细胞提取总RNA,经RT-PCR扩增并制备人IgG 的Fc基因片段,并克隆于已构建好的pBAD-scFv原核表达载体中,转化大肠杆菌Top10,经阿拉伯糖诱导表达融合蛋白scFv-Fc-所表达的可溶性蛋白经亲和层析纯化-SDS-PAGE-Western blot分析鉴定,并用ELISA检测抗体效价-结果:序列分析表明重组质粒pBAD-scFv-Fc基因序列正确;SDS-PAGE分析表明,scFv-Fc融合蛋白分子量为60 ku,且为可溶性蛋白;该蛋白经过His亲和层析纯化-ELISA检测,结果表明,该融合蛋白能够与抗原分子Met特异性结合-结论:改造后的抗体融合蛋白scFv-Fc能与人Met特异性结合,增加了抗体蛋白溶解度,有利于抗体的大量制备-

    Abstract:

    Objective:To generate an anti-Met single-chain antibody fragment(scFv)and human IgG Fc fusion protein and increase the solubility of the scFv. Methods:The human IgG Fc gene was amplified by RT-PCR and cloned into the expression vector of pBAD-scFv which had been constructed previously. The anti-Met scFv-Fc expressing vector was transferred into E.coli. Top10 and the fusion protein expression was induced by L-arabinose.The soluble protein was purified by His-affinity chromatography and characterized by SDS-PAGE and Western blot. The specificity of the scFv-Fc fusion protein was confirmed by ELISA. Results:DNA sequence result showed that the cloned scFv-Fc gene sequence was correct, corresponding to the data of GenBank. SDS-PAGE and Western blot showed that the molecular weight of the fusion protein was about 60 ku.The ELISA results confirmed the scFv-Fc fusion protein could bind to Met protein specifically. Conclusion:The solubilty of scFv-Fc fusion protein was increased when compared with that of the scFv fragment.

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熊 林,张爱霞,李芸茜,张大为,曹伯良,朱 进,唐荣才.人源抗Met基因工程抗体scFv的改造与特性分析[J].南京医科大学学报(自然科学版),2009,29(5):605-608617

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  • 收稿日期:2009-01-03
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