人α型干扰素家族定向进化文库的构建及细胞筛选  

Construction and Screening of the Directed Evolution Library of Interferon-α Family Genes

作　　者：段招军[1] 赵军[1] 王红艳[2] 魏开坤[1] 吕宏亮[1] 李武平[1] 李玉英[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室,北京100052 [2]安徽医科大学

出　　处：《病毒学报》2001年第2期172-174,共3页Chinese Journal of Virology

摘　　要：Mixtures of twelve human interferon alpha genes were digested randomly with DNase I. DNA fragments of 30～50 bps were reassembled into a full length interferon gene with sexual PCR without primer (DNA Shuffling). This primerless PCR product was further amplified with additional PCR with primers containing restriction enzyme sites. Then these PCR products were cloned into the phagemid expression vector PcantAb5E to form an expression library displaying interferon molecules. Regarding WISH cells as a standard strain rich in IFN receptor complex, we developed a new and feasible panning method with WISH cells and performed a competitive washing selection strategy. Using this method in combination with antiviral activity assay, we got two clones showing higher antiviral activity compared with phage IFN α2b. Sequencing results showed that they are hybrids containing two and four interferon alpha genes. Now we are cloning them into prokaryotic expression vector and want to get purified sample to determine their biological activity and biochemistry characters.Mixtures of twelve human interferon alpha genes were digested randomly with DNase I. DNA fragments of 30～50 bps were reassembled into a full length interferon gene with sexual PCR without primer (DNA Shuffling). This primerless PCR product was further amplified with additional PCR with primers containing restriction enzyme sites. Then these PCR products were cloned into the phagemid expression vector PcantAb5E to form an expression library displaying interferon molecules. Regarding WISH cells as a standard strain rich in IFN receptor complex, we developed a new and feasible panning method with WISH cells and performed a competitive washing selection strategy. Using this method in combination with antiviral activity assay, we got two clones showing higher antiviral activity compared with phage IFN α2b. Sequencing results showed that they are hybrids containing two and four interferon alpha genes. Now we are cloning them into prokaryotic expression vector and want to get purified sample to determine their biological activity and biochemistry characters.

关 键 词：DNA SHUFFLING 噬菌体表面呈现 人α型干扰素 文库 

分 类 号：R394.8[医药卫生—医学遗传学] Q78[医药卫生—基础医学]