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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 library of mouse phage antibody with a capacity of 1.7 × 10 8 was successfully constructed and screened out from it. First, total RNA was extracted from the spleens of non-immunized mice, mRNA was isolated and its total cDNA was synthesized by reverse transcription. Subsequently, the VH and VL gene fragments of the antibody heavy and light chain variable regions were respectively amplified by PCR. The two fragments were linked together by the linker of a (Gly4 Ser) 3 pentadecapeptide by recombinant PCR and cloned into The phagemid pCANTAB 5E was electroporated into E. coli TG1. All recombinant phage were recovered by superinfection with the helper phage M 13KO7, which was phage antibody library. N-peptide is a newly discovered neuropeptide whose N-terminal is highly homologous to opioid peptide and interacts with opioid receptor. Its research may help to understand the mechanism of addiction and has clinical value. The biotinylated N-peptide interacted with the antibody library. The streptavidin-loaded magnetic beads were used to enrich the recombinant phage bound to the N-peptide to obtain the higher affinity scFv by four rounds of panning.