小鼠噬菌体抗体库的构建和N-肽结合单链抗体的筛选

Construction of Murine Phage Antibody Library and Selection of N-peptide-binding Single-chain Antibodies

成功构建一库容量为 1.7× 10 8的小鼠噬菌体抗体库 ,并从中淘选出对N 肽有结合活性的单链抗体。首先从未免疫小鼠的脾脏中提取总RNA ,分离出mRNA ,经逆转录合成其总cDNA。随后通过PCR分别扩增出抗体重链和轻链可变区VH、VL 的基因片断 ,再经重组PCR将两片断由一编码 (Gly4 Ser) 3十五肽的接头连在一起 ,并克隆到噬菌质粒 pCANTAB 5E中 ,电击转化大肠杆菌TG1。经辅助噬菌体M 13KO7超感染回收全部重组噬菌体 ,此即噬菌体抗体库。N 肽是一种新发现的神经肽 ,其N端与阿片肽高度同源 ,可与阿片肽受体相互作用。对其研究可能有助于了解成瘾机制和有临床应用价值。将生物素化的N 肽与抗体库相互作用 ,用偶联有链霉亲和素的磁珠富集与N 肽结合的重组噬菌体 ,经四轮淘选 ,得到亲和力较高的单链抗体。

A murine phage antibody library containing 1.7×10 8 independent clones was constructed and affinity panning of anti\|N\|peptide antibody fragments was carried out. First,mRNA was purified from the total RNA extracted from fresh spleens of nonimmunized mice of Kunming,then the cDNA library was achieved via reverse transcription PCR.Gene fragments encoding V\-H and V\-L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues (Gly\-4Ser)\-3 through primary PCR. And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB 5E vector and introduced into E.coli TG1 by means of electroporation. The phagemid particles displaying functional ScFvs were rescued by reinfection of helper phage M13KO7,thus a murine antibody library was obtained. One N\|peptide\|binding ScFv clone was selected from the phage antibody library using affinity panning. The target antigen, N\|peptide was biotinylated using photobiotin first. Then the biotin\|labelled N\|peptide interacted with the phage antibody library. The streptavidin paramagnetic beads were added subsequently to bind the biotinylated N\|peptide\|specific phage particle complex. Washing,elution and reinfection were ensued sequentially. After 4 rounds panning, one clone was verified to show higher affinity to N\|peptide by dot blot.