基于计算生物学方法的小容量单域抗体库的构建及食品中雌二醇特异性抗体的筛选  

Construction of Small Single Domain Antibody Library Based on Computational Biology and Screening of Anti-estradiol Antibody

作　　者：刘超[1] 曲晓辰 王冲[1] 宁保安[1] 高志贤[1] 

机构地区：[1]军事医学科学院卫生学环境医学研究所天津市环境与食品安全风险监控技术重点实验室,天津300050

出　　处：《解放军预防医学杂志》2016年第3期308-311,共4页Journal of Preventive Medicine of Chinese People's Liberation Army

基　　金：国家重大科学仪器设备开发专项(No.2013YQ14037106);国家自然科学基金(No.81472985;No.21207161);国家科技支撑计划项目(No.2012BAK08B06)

摘　　要：目的构建骆驼源单域抗体实体库和虚拟库,为快速筛选检测食品中小分子污染物的特异性抗体提供新方法。方法通过基因克隆、一代测序、同源性分析的方法构建骆驼源单域抗体基因库,通过二级结构分析和同源建模的方法建立单域抗体的三维结构虚拟库,利用分子对接技术实现雌二醇特异性抗体的虚拟筛选,并以雌二醇小分子为靶标进行初步验证。结果构建的小容量单域抗体库含有2000株抗体,每一株抗体的遗传背景、蛋白质结构及抗体抗原互补决定区(CDR)的信息清晰;通过分子对接得到结合雌二醇小分子能力最高的一株抗体的半经验结合自由能绝对值为9.56;相互作用分析结果显示,单域抗体结构中和雌二醇小分子发生相互作用的主要为3个CDR区形成的loop结构,经间接ELISA验证,其和雌二醇结合的50%抑制率(IC50)值为20 ng/ml,检测限(LOD,IC10)为4.097 ng/ml。结论通过计算生物学和基因工程技术相结合的方法构建的骆驼源单域抗体库,针对食品中常见的激素类兽药雌二醇,可以筛选得到背景清晰、具有较高亲和力的特异性抗体。Objective To provide new method for screening the specific antibody against small molecular pollutant in food by constructing camelid single domain antibody （VHH） physical library and virtual library. Methods The single domain antibody gene library was constructed by gene cloning, sequencing, homology analysis, the 3D structure virtual library was constructed by secondary structure analysis and homology modeling, and the molecular docking analysis was used to screen the anti-estradiol （E2） antibody in silico. Results The constructed small single domain antibody library contained 2000 antibody strains, and the genetic background, protein structure and the information of complementarity determining region （CDR） of every antibody strain was clear. The binding affinity of the best anti-estradiol VHH screened by molecular docking was 9.56. The dedicated domain of VHH interacted with estradiol was found in the loop structure of CDR. The 50% inhibition concentration （IC50） for E2 was 20 ng/ml, and the limit of detection （ LOD, IC10） was 4.097 ng/ml tested by indirect ELISA. Conclusion The camelid single domain antibody library was constructed by computational biology and genetic engineering techniques from which the specific VHH against E2 was screened with clear background and relatively high affinity.

关 键 词：单域抗体 抗体库技术 计算生物学 雌二醇 

分 类 号：R392.11[医药卫生—免疫学]