中文摘要：

目的构建并表达绿色荧光蛋白（GFP）和鼠抗人大肠癌单链抗体ND-1scFv的融合蛋白,产生具有荧光的抗体。方法将鼠抗人大肠癌单链抗体ND-1scFv的基因克隆到pET28a（＋）-GFP的表达载体,转化到大肠杆菌BL21中进行融合基因ND-1-scFv/GFP诱导表达。Ni-NTA亲和层析对表达产物进行分离、纯化,免疫印迹和荧光显微镜方法验证融合蛋白的表达。结果 ND-1scFv被克隆到表达载体pET28a（＋）-GFP中,诱导表达的融合蛋白以包涵体形式存在,分子量为58kDa。SDS-PAGE灰度扫描结果显示纯化后的蛋白纯度为90%,荧光显微镜检测显示,表达有目的蛋白的大肠杆菌BL21具有明显的绿色荧光。结论成功构建并表达融合基因ND-1-scFv/GFP,为该单抗的肿瘤特异成像研究奠定了基础。E.coli

英文摘要：

Objective To study the construction and expression of the recombinant ND-1scFv/GFP,a fusion protein of green fluorescent protein（GFP）and single-chain variable fragment of monoclonal antibody ND-1 against human clolorectal carcinoma to produce functional antibodies with fluorescent labels.Methods ND-1 scFv gene was subcloned into pET28a（＋）-GFP vector and the expression of the fusion protein was induced in BL21 by IPTG.Ni-NTA resin affinity chromatography was used to isolate and purify the protein product.The purified ND-1-scFv/GFP protein was examined by SDS-PAGE and fluorescence microscope.Results SDS-PAGE analysis revealed that ND-1-scFv/GFP fusion protein was expressed in the form of inclusion bodies with a molecular weight of 58 kDa,grayscale scanning showed that the purity of purified ND-1scFv/GFP was over 90%.The strong green fluorescence in BL21 indicates that the fusion protein was functionally expressed.Conclusion Our successful construction and expression of scFv-GFP fusion gene is helpful for the further application of ND-1-scFv in tumor-targeted imaging.