引入易错PCR和DNA-shuffling技术构建单链抗体库  

The introduction of error-prone PCR and DNA-shuffling technology to build mono-chain antibody library

作　　者：杨浩[1] 杨青平 查成喜[1] 韩跃武[1] 

机构地区：[1]兰州大学基础医学院 [2]甘肃省第三人民医院内分泌科,甘肃兰州730000

出　　处：《基础医学与临床》2012年第1期56-60,共5页Basic and Clinical Medicine

摘　　要：目的引入易错PCR和DNA-shuffling技术构建高库容量的单链抗体库。方法收集不同年龄、性别、健康状态的人的静脉血各5 mL,提取单个核细胞总RNA,反转录为cDNA,PCR扩增VH和VL基因,应用易错PCR对VH和VL基因进行突变,同时用DNA-shuffling技术对全长单链抗体基因进行体外改组,获得的分子与T载体连接,转化E.coli DH5α,选取阳性克隆,经菌落PCR后酶切鉴定抗体库多样性,计算分析单链抗体库容量。结果成功构建了库容量为4.37×1013的单链抗体库,经酶切鉴定,初步判定抗体库多样性良好。结论引入易错PCR和DNA-shuf-fling技术,成功构建了单链抗体库,为后续筛选高亲和力抗体奠定了实验基础。Objective To construct a mono-storage capacity single-chain PCR and DNA-shuffling technologies. Methods Peripheral blood was antibody （scFv） library using error-prone collected from different age, gender and healthy people. Total RNA was extracted from human peripheral blood mononuclear cells and reversely transcribed to cDNA by RT-PCR. VH and VL genes were amplified by PCR and mutated by error-prone PCR. The full-length ScFv fragments were recombinated with the DNA-shuffling technology in vitro, and then connected with T vector and transformed into E. coli DH5α. Antibody library diversity was identified by restriction enzyme digestion after colony PCR, and then the capacity of scFv was calculated. Results ScFv library was constructed by error-prone PCR and DNA-shuffling with high-storage capacity and genetic diversity has been constructed, finity antibodies. The 4. 37 × 10^13 capacity and better diversity techniques. Conclusions The scFv library which laid a foundation of selection high-affinity antibodies.

关 键 词：易错PCR DNA-shuffling 单链抗体库 

分 类 号：R392.11[医药卫生—免疫学]