利用 PCR 扩增及基因合成构建 Adnectin半随机核糖体展示文库

摘要

目的：构建Adnectin半随机的核糖体（ mRNA）展示文库。方法分析Adnectin核糖体展示文库结构序列的编码氨基酸序列，利用无意突变建立酶切位点，使用PCR扩增和基因合成两种方法相结合，构建Adnectin半随机核糖体展示文库，通过限制性内切酶及DNA测序证实序列正确性，对文库转化菌的滴定和插入失活蓝白菌斑筛查和计数测定计算文库的库容。结果经测序证实文库结构的正确性，文库的库容为1．54×1013／mL。结论成功构建Adnectin半随机的核糖体展示文库，方法简单，库容量大，该文库的建成为亲和各种相关蛋白的Adnectin结合序列奠定基础。%Objective To construct semi-random Adnectin ribosome display library .Methods Adnectin ribosome display library was constructed by PCR amplification combining with gene synthesis method .PCR products contained the restrictive sites of SpeⅠbased on nonsense mutation .The recombinant plasmids were identified by restriction endonuclease enzyme analysis and DNA sequence .The positive recombinant clones were screened and library capacity was measured by blue-white test.Result The library was confirmed by sequencing and library capacity was 1.54 ×1013/mL.Conclusions To corstruct successful Adnectin semi-random ribosomes display library ( mRNA) used simple and easy method .The built library laid the foundation for Adnectin affinity with various related protein .