文章摘要
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,2009,29(5):605~608617
人源抗Met基因工程抗体scFv的改造与特性分析
Reconstitution of human anti-Met genetic engineering antibody scFv
投稿时间:2009-01-03  
DOI:10.7655
中文关键词: Met  基因工程抗体  融合蛋白  Fc片段
英文关键词: Met  genetic engineering antibody  fusion protein  Fc fragment
基金项目:江苏省社会发展项目资助(BS2007019);南京军区“122”工程资助
作者单位
熊 林 南京医科大学病理系,卫生部抗体技术重点实验室,江苏 南京 210029 
张爱霞 南京医科大学病理系,卫生部抗体技术重点实验室,江苏 南京 210029 
李芸茜 南京医科大学病理系,卫生部抗体技术重点实验室,江苏 南京 210029 
张大为 南京医科大学病理系,卫生部抗体技术重点实验室,江苏 南京 210029 
曹伯良 南京医科大学病理系,卫生部抗体技术重点实验室,江苏 南京 210029 
朱 进 南京军区军事医学研究所,江苏 南京 210002 
唐荣才 江苏省血液中心,江苏 南京 210037 
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中文摘要:
      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?
英文摘要:
      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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