利用IgA蛋白酶β结构域为骨架构建细菌表面展示随机肽库

Construction of a random peptide library displayed on the bacterial surface using theβ-domain of IgA protease as an anchoring motif

以IgA蛋白酶β结构域为骨架构建细菌表面展示随机肽库。设计合成两条寡核苷酸双链,链1含有一个随机编码序列(NNK)10,其3'端可与链2互补。通过两条链的退火、延伸、酶切、回收后与酶切、去磷酸化的载体以最佳连接比连接,采用酚∶氯仿抽提去除残留的连接酶,分批进行电转化,获得一库容量为5×106的随机肽库。随机挑选10个克隆进行测序,结果显示所构建肽库的基本框架与预期设计相符,四个寡核苷酸出现的频率也与理论值相接近,表明所构建的随机肽库是成功的。

Theβ-domain of IgA protease was used as a carrier protein to construct a bacterial surface display li-brary.Two oligonucleotides were synthesized.Oligo1contained a random oligonucleotide encoding region(NNK) 10 and its3'-end was complement to oligo2.The two synthesized oligonucleotides was annealed and then extended by Klenow Fragment.The double-stranded oligonucleotides was digested by SfiⅠand purified by PAGE,then inserted into the digested,dephosphorylated vector at the best ligation ratio V:I.The ligation product was purified by phenol-chloroform extraction and electroporated into XL-1.Finally,a library with diversity5×10 6 was obtained.The results of10sequenced clones revealed that the basic frame of the constructed library was correct and the four bases of A/T/G/C are distributed randomly.