全合成人源性噬菌体抗体库的构建

Construction of a fully synthetic human phage display antibody library

目的:构建全合成人源性噬菌体抗体库。方法:选择部分使用频率较高的人抗体胚系基因家族的框架区基因进行人工合成,同时人工设计合成半随机CDR3,通过重叠拼接延伸PCR的方法合成抗体基因。然后利用限制性内切酶SfiⅠ、NotⅠ分别双酶切抗体基因和噬菌体展示载体。连接后的重组噬粒通过电击转化的方法转入大肠杆菌TG1,构建抗体库。结果与结论:PCR结果显示,通过优化后的方法能在保证抗体库多样性的前提下,高效地获得抗体基因。经过数十次电击转化构建了库容量为2×109的全合成抗体库,菌落PCR、测序及筛选结果表明,抗体库质量优良,为筛选人源性治疗抗体奠定了基础。

Objective：To construct a fully synthetic human phage display antibody library. Methods：The frameworks of human antibody germline genes and semi-random CDR3s （complementarity determining region 3 ） were synthesized artificially. The antibody genes were synthesized by SOE-PCR（ splicing overlap extension PCR）. Then the antibody genes and the phage display vector were digested by restriction endonucleases Sfi Ⅰ and Not Ⅰ. The recombinant phagemid was electroporated into E. coli TG1. Results and conclusion：As long as the diversity of antibody library was ensured, the antibody genes could be obtained expediently by the improved method. A fully synthetic human phage display antibody library of 2 × 10^9 was constructed through numerous electro-transformations. The quality of the library was proven to be fine by colony PCR and sequencing. It established a base for screeniw, human theraneutic antibody.