Lipocalin核糖体展示文库的构建及雌二醇特异性模拟抗体的筛选

Construction of Ribosome Displayed Library Based on the Lipocalin Scaffold and Screening Anticalins to Estradiol

[目的]构建Lipocalin核糖体展示文库,并筛选雌二醇特异性模拟抗体。[方法]以Lipocalin蛋白家族中的BBP(胆汁三烯结合蛋白)为模板,设计包含16个随机突变位点的引物,合成BBP基因文库;引入核糖体展示所必需的全部组件,采用重叠PCR技术将BBP库与核糖体展示组件进行拼接,构建核糖体展示Anticalin模拟抗体库;再将构建的Anticalin文库进行体外转录与翻译,产生模拟抗体-核糖体-mRNA三联复合体文库。以小分子抗原雌二醇为靶标,采用固相亲和方法筛选抗雌二醇抗体,通过改变Mg2+浓度将模拟抗体-核糖体-mRNA三联复合体解离,得到与模拟抗体对应的mRNA,经过RT-PCR得到筛选后的DNA文库并重新引入核糖体展示元件进行下一轮淘筛。[结果]通过5轮生物淘筛,模拟抗体得到富集,获得高特异性抗雌二醇抗体。其中有一株克隆的亲和力为54 mmol/L(Kon=4.975 6×104个/M·S;Koff=0.002 7个/S)。IC50与LOD值分别为50和0.071 ng/ml。[结论]获得的雌二醇模拟抗体可以作为抗体并用于检测动物体内雌二醇的残留。

[ Objective ] To construct Lipoealin ribosome display library and screen out estradiol specific simulation antibody. [ Method ] The bilin - binding protein （ BBP）, a member of the fipoealin family from Pieris brassieae, was employed as a model system for the preparation of a random library. In order to specifically complex estradiol （E2）, a steroid ligand with extensive medical function to animals and poultries, 16 amino acid residues at the center of the binding site, which is formed by four loops on top of an eight - stranded β - barrel, were subjected to targeted random mutagenesis. From the resulting library the estradiol - binding BBP variants - termed ＇ anticalins＇ exhibiting binding activity for compounds like estradiol, have been selected by combined use of ribosome display and screening techniques. [ Result] Four variants were identified that complex estradiol with high affinity, bound estradiol specifically among them and its 50% inhibition concentration （ICso） for E2 was 50 ng/ml and the limit of detection （ LOD ： IC50 ） was 0.071 ng/ml after optimization of reaction conditions. [ Conclusion ] Our findings suggest that anticalin can be used to detect estradiol of animal.