BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filame...BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.展开更多
目的探讨以包涵体形式表达的抗鼻咽癌单克隆抗体(mAb)BAC5的单链抗体(BAC5-scFv)的纯化、复性方法,并对其活性进行检测。方法扩增pET-22b-scFv质粒转化的大肠杆菌BL21(DE3)菌株,培养、破菌后,分离和变性包涵体,用Ni-NTA His Bind层析柱...目的探讨以包涵体形式表达的抗鼻咽癌单克隆抗体(mAb)BAC5的单链抗体(BAC5-scFv)的纯化、复性方法,并对其活性进行检测。方法扩增pET-22b-scFv质粒转化的大肠杆菌BL21(DE3)菌株,培养、破菌后,分离和变性包涵体,用Ni-NTA His Bind层析柱纯化变性的scFv。经稀释、透析及尿素梯度凝胶层析柱3种方法进行复性。用细胞免疫组化染色和蛋白印迹法(Western blot),鉴定复性后的BAC5-scFv的免疫活性。结果Ni-NTA His Bind亲和层析柱能有效纯化变性的scFv。以尿素梯度凝胶层析柱复性的蛋白回收率最高。免疫细胞化学染色法检测及Western blot分析证实,复性后的BAC5-scFv可与CNE2细胞上的抗原特异性结合。结论以包涵体形式表达的BAC5-scFv经变性、纯化及复性后,获得良好的免疫活性,为大量制备具有活性的BAC5-scFv,并用于鼻咽癌的放射免疫显像和治疗研究奠定了基础。展开更多
文摘BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.
文摘目的探讨以包涵体形式表达的抗鼻咽癌单克隆抗体(mAb)BAC5的单链抗体(BAC5-scFv)的纯化、复性方法,并对其活性进行检测。方法扩增pET-22b-scFv质粒转化的大肠杆菌BL21(DE3)菌株,培养、破菌后,分离和变性包涵体,用Ni-NTA His Bind层析柱纯化变性的scFv。经稀释、透析及尿素梯度凝胶层析柱3种方法进行复性。用细胞免疫组化染色和蛋白印迹法(Western blot),鉴定复性后的BAC5-scFv的免疫活性。结果Ni-NTA His Bind亲和层析柱能有效纯化变性的scFv。以尿素梯度凝胶层析柱复性的蛋白回收率最高。免疫细胞化学染色法检测及Western blot分析证实,复性后的BAC5-scFv可与CNE2细胞上的抗原特异性结合。结论以包涵体形式表达的BAC5-scFv经变性、纯化及复性后,获得良好的免疫活性,为大量制备具有活性的BAC5-scFv,并用于鼻咽癌的放射免疫显像和治疗研究奠定了基础。
