抗癌胚抗原纳米抗体展示文库的构建与筛选

Construction and Identification of Nanobody Display Libraries Recognizing Arcinoembryonic Antigen

利用噬菌体展示技术进行纳米抗体库的构建和筛选,获得抗癌胚抗原(CEACAM5)的高亲和力、强特异性纳米抗体,为相关癌症诊断及靶向治疗的应用奠定基础。使用重组CEACAM5抗原免疫羊驼,分离免疫后羊驼的外周血淋巴细胞,提取总RNA后通过RT-PCR技术扩增羊驼重链抗体可变区(VHH)片段,构建纳米抗体文库。采用噬菌体展示技术和固相淘选方法,筛选得到强阳性克隆,经大肠杆菌表达和镍离子亲和层析获得纳米抗体,最终利用Biacore分析其亲和力和特异性。通过3轮的淘选,获得一株纳米抗体亲和力达到2.6×10-10mol·L-1,对同源性蛋白CEACAM-1、-3、-6、-8及肿瘤蛋白AFP均无交叉反应性。利用噬菌体展示技术成功获得了抗癌胚抗原(CEACAM5)的高亲和力、强特异性纳米抗体,可用于后续相关癌症诊断和靶向治疗。

To construct nanobody library,and to screen it,high affinity and strong specificity nanobody were obtained to arcinoembryonic antigen(CEACAM5)using phage display technology,provide a substantial basis for the application of related cancer diagnosis and targeted therapy.Peripheral blood lymphocytes of alpacas immunized by recombinant CEACAM5 antigen were separated,followed by the extraction of total RNA.The variable heavy chain antibody(VHH)fragments of alpaca were amplified by RT-PCR to construct a nanobody library.After rescue and amplification of helper phage from the immune library,strong positive clones were obtained by panning.Nanobody was selected to express in E.coli and purified by nickel ion affinity chromatography.Finally,the affinity and specificity of the nanobody were analyzed by Biacore.After three rounds of phage selection,a nanobody,named Nb5,was selected and showed the affinity of 2.6×10-10 mol·L-1,and there was no cross-reactivity to homologous proteins CEACAM-1,-3,-6,-8 and tumor protein AFP.The high affinity and strong specificity of anti-carcinoembryonic antigen(CEACAM5)nanobody was successfully obtained by phage display technology,which pave the way for the application of related cancer diagnosis and targeted therapy.