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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
Objective To construct and express a fusion protein of green fluorescent protein (GFP) and ND-1 scFv, a murine anti-human colorectal cancer single chain antibody, to produce a fluorescent antibody. Methods The murine anti-human colorectal cancer ND-1 scFv gene was cloned into pET28a (+) - GFP expression vector and transformed into Escherichia coli BL21 for inducing the fusion gene ND-1-scFv / GFP expression. Ni-NTA affinity chromatography on the expression product was isolated, purified, Western blot and fluorescence microscopy to verify the expression of the fusion protein. Results The ND-1scFv was cloned into the expression vector pET28a (+) - GFP. The fusion protein was expressed as inclusion bodies with a molecular weight of 58 kDa. SDS-PAGE gray scale scanning showed that the purity of the purified protein was 90%. Fluorescence microscopy showed that E. coli BL21 expressing the target protein had obvious green fluorescence. Conclusion The successful construction and expression of the fusion gene ND-1-scFv / GFP laid the foundation for the study of tumor-specific imaging of this monoclonal antibody. E. coli