一种新型转铁蛋白受体结合肽的筛选与鉴定

目的以转铁蛋白受体(TfR)为靶标,从噬菌体随机七肽库淘选TfR结合肽并进行鉴定。方法经过三轮筛选,从平板上挑取分隔良好的特异性TfR结合肽单克隆,采用ELISA初步鉴定后行扩增测序及多肽合成,采用流式细胞仪检测人肝癌HepG2细胞表面TfR表达、采用免疫荧光实验及激光共聚焦实验行噬菌体与细胞共定位鉴定、采用短肽—噬菌体竞争抑制实验检测短肽对噬菌体的竞争抑制率,以正常肝脏L-O2细胞为对照。结果特异性TfR结合肽克隆最终富集度达80倍;初步检测到20个阳性克隆,其中9号克隆对TfR亲和力最高,测序表明其展示短肽序列为AHLHNRS,以碱性氨基酸为主,经HPLC检测纯度为99.91%;HepG2细胞表面TfR表达显著高于L-O2细胞;免疫荧光实验及激光共聚焦实验均显示9号噬菌体(P9)出现在HepG2细胞表面,而对照L-O2细胞无此现象;短肽与噬菌体P9对TfR的结合存在竞争关系,并且呈浓度依赖性,而野生型噬菌体M13与L-O2不存在此现象。结论本研究获得一条与TfR具有特异性结合能力的TfR结合肽;此为进一步构建肿瘤和脑源性疾病靶向治疗药物奠定了基础。

Screening and identification of a novel transferrin receptor binding peptide

Objective To select TfR(transferring receptor)-binding peptide from phage random display library,using TfR as the target.Methods After three rounds of screening,some positive clones were picked and analyzed by competitive ELISA.The positive clones were sequenced and the peptide was synthesized according to the sequences.The TfR of the HepG2 was analyzed by flow cytometer.Phages co-localized with HepG2 cells were detected by immunofluorescence microscopy and confocal laser scanning microscope.The ratio of competitive binding of the peptide virsus phage was established by competitive inhibition experiments,the heathy liver cell L-O2 was taken as control.Results The phage display library was enriched 80 more folds;twenty positive clones were found,of which No.9(designated P9) had the highest affinity to TfR,the sequences was AHLHNRS,mainly consisted of basic amnio acids,HPLC detection showed the purity degree was 99.91%;the TfR expression in HepG2 was more higher than that in the control cell L-O2;the results of both immunofluorescence microscopy and confocal laser scanning microscope showed the phage P9 was round HepG2,but L-O2 not;the results of competitive inhibition manifested P9 competitively binded hepatoma HepG2 in concentration dependent manner,but the wild type M13 and L-O2 not.Conclusion A peptide that bind to TfR specially can be successfully got,which may provide a basis for anti-tumor drugs and targeting brain disease drugs.