大肠杆菌表面CS3菌毛展示随机肽库的构建

Construction of a CS3 fimbria-displayed random peptide library on Escherichia coli cell surface

目的:在肠毒素性大肠杆菌CS3菌毛表面构建 10肽随机肽库．方法:首先将原有的单酶切CS3菌毛呈现载体改造为双酶切载体,并证实改造后的载体能正确形成CS3菌毛．同时设计合成2条寡核苷酸序列,链1含有1个10肽随机编码序列(NNS)10, 链2可与链1的3′端互补．两条链经过退火、延伸、酶切和回收与经过同样酶切的呈现载体连接,连接产物纯化后分多次电击转化,获得随机肽库．随机挑选10个克隆进行测序并对测序结果进行分析．结果:获得1个库容量为1．8×106大小的随机肽库．测序结果显示,所构建肽库的基本框架与预期设计相符,4个寡核苷酸出现的频率也与理论值相接近．结论:在肠毒素性大肠杆菌CS3菌毛表面成功构建库容量为1．8×106大小的10肽随机肽库．为下一步利用肽库进行筛选奠定了基础．

AIM： To construct a CS3 fimbria-displayed random peptide library on the Escherichia coli cell surface. METHODS： Firstly, a new display vector with double restriction sites was reconstructed, and the ability of the new vector to form CS3 fimbriae on E. coli surface was confirmed by Western blot and transmission electron microscopy. Then two oligonucleotides were synthesized, one of which contained a random oligonucleotide encoding region （NNK）10 and the other was its complement. The two synthesized oligonucleotides was annealed and then extended by Klenow Fragment. The double-stranded oligonucleotides were digested by Xho Ⅰ and BamH Ⅰ and purified by PAGE, then inserted into the digested display vector. The ligation product was purified and electroporated into XL1-Blue. Ten randomly selected clones were sequenced and the sequences were analyzed.RESULTS： A library with diversity of 1.8 × 10^6 was obtained. The sequencing results confirmed the basic frame of the constructed library to be correct and the bases of A, T, G, and C were distributed randomly.CONCLUSION： A random ten-peptide library is displayed on the Escherichia coli cell surface using CS3 fimbriae display system.