中国农业科学院农业信息研究所 中国农学会
Agricultural Information Institute of CAAS China Association of Agricultural Science Societies
En
Ch
中国农业科学院农业信息研究所 中国农学会 主办

AI检索

订阅

返回顶部
当前位置: 首页 > 文章 > 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 安徽农业科学 2013 (31) 12251-12254+12261
Position: Home > Articles > Construction of Ribosome Displayed Library Based on the Lipocalin Scaffold and Screening Anticalins to Estradiol Journal of Anhui Agricultural Sciences 2013 (31) 12251-12254+12261

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

作  者：
刘建青;宁保安;高志贤
单  位：
包头师范学院生物科学与技术学院;天津市卫生学环境医学研究所
关键词：
雌二醇;BBP;核糖体展示;突变文库
摘  要：
[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。
译  名：
Construction of Ribosome Displayed Library Based on the Lipocalin Scaffold and Screening Anticalins to Estradiol
作  者：
LIU Jian-qing;School of Biological Science and Technology,Baotou Normal University;
关键词：
Estradiol(E2);;Bilin-binding protein(BBP);;Ribosome display;;Mutant library
摘  要：
[Objective] To construct Lipocalin ribosome display library and screen out estradiol specific simulation antibody.[Method] The bilin- binding protein(BBP),a member of the lipocalin family from Pieris brassicae,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(IC50) for E2 was 50 ng/ml and the limit of detection(LOD:IC10) was 0.071 ng/ml after optimization of reaction conditions.[Conclusion]Our findings suggest that anticalin can be used to detect estradiol of animal.

相似文章

导出引用
计量
文章访问数: 9
HTML全文浏览量: 0
PDF下载量: 0

所属期刊

推荐期刊

友情链接Friendly link
农业信息研究所Agricultural information institute 农业智能知识服务平台Agricultural intelligent knowledge service platform 农业学术服务平台Agricultural Knowledge Service System 中国知网CNKI 维普网VIP 万方WANFANG DATA
联系我们Contact us

电话Tell：010-82109652-808

邮箱E-mail：agrijournal@caas.cn

服务Service
关于我们About us 意见建议Opinions and suggest
版权说明Copyright
单位：中国农业科学院农业信息研究所 中国农学会 © 2017中国农业科学院农业信息研究所 ICP备案号：京ICP备09089781号-46