癌胚抗原单链抗体在细菌表面的展示及其应用

Display of the Carcinoembryonic Antigen scFv on the Bacterial Surface and Its Application

癌胚抗原(carcinoembryonic antigen,CEA)作为肿瘤标志物,在临床肿瘤诊断及肿瘤靶向治疗等方面具有重要应用价值。癌胚抗原单链抗体(CEA-specific single chain antibody fragments,CEA-scFv)能够特异性结合癌胚抗原。本研究将癌胚抗原单链抗体展示于大肠杆菌细胞表面,分析其作为癌胚抗原检测平台和细菌靶向载体的可行性。首先将CEA-scFv基因克隆到表面展示载体pBAD-OmpA-m Cherry中,经酶切和测序证实,成功构建了重组质粒pBAD-OmpA-mCherry-CEA。重组菌经阿拉伯糖诱导后可检测到红色荧光,荧光强度在胰酶的作用下降低,全菌ELISA检测呈阳性反应,提示融合蛋白和目的蛋白质在重组菌表面展示成功。由Western印迹分析可知,融合蛋白质的相对分子质量约为85 k D,符合预期设计。进一步的研究提示,重组菌在大肠杆菌表面展示的癌胚抗原单链抗体具有生物活性,能够有效结合A549细胞裂解液的癌胚抗原。在细菌侵染细胞实验中,与对照组相比,重组菌孵育A549细胞后细胞内可明显观察到点状红色荧光,证明重组菌能够靶向侵入癌胚抗原阳性肿瘤细胞。本研究为癌胚抗原相关的快速诊断和基于细菌载体的肿瘤靶向治疗奠定了实验基础。

As a tumor marker,the carcinoembryonic antigen（ CEA） has important clinical application values in tumor diagnosis and tumor-targeting therapy. CEA-specific single chain antibody fragments（ CEA-scFv） specifically bind to CEA. In the present study,CEA-scFv was displayed on the surface of Escherichia coli（ E. coli）,and its feasibility as a detection platform for CEA and bacterial targeting vectors was analyzed. Firstly the CEA-scFv gene was cloned into the surface display vector p BAD-Omp Am Cherry,and the recombinant plasmid p BAD-Omp A-m Cherry-CEA was successfully constructed,which was confirmed by restriction enzyme digestion and sequencing. Red uorescence of the recombinant bacteria induced by arabinose can be detected,and the fluorescence intensity was decreased by trypsin digestion. Moreover,the whole-cell ELISA test showed a positive reaction,suggesting that the fusion protein and protein of interest was successfully displayed on the surface of the recombinant bacteria.Western blotting analysis showed that the relative molecular mass of the fusion protein was about 85 k D,which was in line with the expectation. Further research suggested that CEA-scFv displayed on the surface of the recombinant bacteria had biological activity,and bound the CEA of A549 cell lysates effectively. Compared with the control group,the punctate red fluorescence was clearly observed in recombinant bacteria-infected A549 cells,indicating that the recombinant bacteria can target and invade CEA-positive tumor cells. This study provides an experimental basis for CEA-related rapid diagnosis and tumor targeted therapy based on bacterial carriers.