【摘要】 目的:用基因工程手段获得有活性的抗独特型抗体I50,并在体外鉴定其活性。方法:以fuse5-I50为模板,用PCR方法扩增出抗独特型抗体I50基因,并将其插入到pET25b(+)中构建原核表达载体pET25b-I50。含有重组质粒pET25b-I50的菌株E.coli BL21(DE3)经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导能够得到有效表达。采用Western印迹鉴定I50蛋白的表达,采用透析复性方法恢复I50蛋白的活性,并用Dot-ELISA法,淋巴细胞增殖实验鉴定其活性。结果:成功的构建了原核表达载体pET25b-I50,并经IPTG诱导获得了I50蛋白,该蛋白以包涵体形式高效表达,经纯化后纯度达90%以上。Western印迹鉴定表达的蛋白相对分子质量约15000,与预期相符。Dot-ELISA法鉴定复性后的蛋白已恢复活性,并能够刺激淋巴细胞的增殖,且呈剂量依赖关系。结论:成功获得了有活性的I50抗独特型抗体,为其应用于鼻咽癌的治疗鉴定了基础。更多还原

【Abstract】 Objective To obtain I50 anti-idiotype antibody and identify its activity in vitro.Methods I50 anti-idiotype(Id) antibody gene was amplified from the template of fuse 5-I50 by PCR to construct a prokaryotic expression vector pET25b-I50.The expression of pET25b-I50 in E.coli BL21(DE3) was induced by isopropylthio-β-D-galactopyranoside(IPTG) and was confirmed by SDS-PAGE and Western blot with Ab1(FC2) monoclonal antibody and an anti-hexahistidine tag antibody.The method of dialysis refolding was used to restore the activity of I50 anti-Id antibody,which was measured by Dot-ELISA and lymphocyte proliferation assay.Results The recombinant vector was successfully constructed and the recombinant protein was successfully expressed and purified with 90% purity.The relative molecular weight of the expressed protein was 15 kD,which was in accordance with expectation.The activity of I50 anti-Id antibody could be restored and could promote the proliferation of lymphocyte in a dose-dependent manner.Conclusion These results suggested that I50 anti-Id protein vaccine is likely an option in the therapy against nasopharyngeal carcinoma in vivo.更多还原