一种用于穿透多肽筛选的随机文库的构建及筛选

以增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)为示踪物，在pET-14b载体上构建编码12个氨基酸的随机多肽表达文库.建立一种简便、经济、有效的文库筛选方法，从所构建的文库中筛选出细胞穿透多肽（cell-penetrating peptide, CPP）. 采用点突变技术，首先在pET-14b载体多克隆位点NdeⅠ和XhoⅠ之间加入4个限制性内切酶位点，随后在BamH Ⅰ位点后加入三联终止密码子，接着再利用亚克隆的方法在Kpn Ⅰ 和XhoⅠ之间插入EGFP，形成一个新的用于原核表达示踪蛋白的载体pET-14bMCStop/EGFP.最后再利用点突变技术在上述构建的示踪载体的多克隆位点XhoⅠ和BamH Ⅰ之间插入36个随机碱基序列.以His-Tat-EGFP作为工具建立有效的筛选方法，利用这种方法对文库进行筛选. 酶切和测序表明，示踪载体的构建是正确的，且在大肠杆菌中可有效地表达出His标记的EGFP.在示踪载体的基础上构建的随机多肽文库至少包含了10⁵个独立克隆，其中90%以上的克隆插入的随机片段都是36个碱基.建立的筛选方法是可行的，并用此方法进行了初步的筛选.

Construction of Random Peptide Library and Screening of Penetrating Peptides

Some proteins and peptides possess the intrinsic property to enter across the cell membrane from the external environment. These peptides are called cell penetrating peptides(CPPs). A screening system based on random peptide libraries has been established for searching new CPPs. The vector pET-14bMCStop/EGFP with 6×His, EGFP and three stop codons were constructed and a random peptide library of twelve amino acids in pET-14bMCStop/EGFP(N)₃₆ were constructed by site directed mutagenesis. DNA sequencing and restriction enzyme digestion analysis showed that the constructed vector pET-14bMCStop/EGFP was correct. The library harbored E.coli BL21(DE3) was large enough to produce 105 independent clones. Sequencing of a random sample of 14 of these clones showed that more than 90% of the plasmids have been inserted with 36 base pairs. No open reading frame shift happened to all these plasmids. Each plasmid of the library was expressed in E.coli BL21(DE3) and purified by using Qiagen Ni²⁺NTA agarose. Purified fusion proteins were added into HeLa cells which are observed under fluorescence microscope. One clone carrying inserted peptide showed the ability to enter the cells across cell membrane.