基于纳米抗体CDR3区抗原表位展示的β-HCG抗体制备及鉴定  

Preparation and identification of anti-β-HCG antibody based on display of antigenic epitopes in CDR3 region of nanobody

作　　者：王蕾[1] 霍景瑞 张国安 贾力 田毅[1] 杨晓晖[2] 张晶晶 刘颖[1] 吴楠 刘英富 WANG Lei;HUO Jing-rui;ZHANG Guo-an;JIA Li;TIAN Yi;YANG Xiao-hui;ZHANG Jing-jing;LIU Ying;WU Nan;LIU Ying-fu(Department of Pathogenic Biology and Immunology, Cangzhou Medical College, Cangzhou 061001, China;Science and Technology Experiment Center, Cangzhou Medical College, Cangzhou 061001, China;Nanobody Technology Innovation Center of Cangzhou)

机构地区：[1]沧州医学高等专科学校病原生物与免疫学教研室,061001 [2]沧州医学高等专科学校科技实验中心,061001 [3]沧州市纳米抗体技术创新中心

出　　处：《天津医药》2019年第8期785-789,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目（11672332,81772018）;天津市自然科学基金（17JCZDJC35400）;沧州市科学技术研究与发展指导计划（162302146,162302158)

摘　　要：目的通过纳米抗体CDR3区展示β-HCGC端表位的方式,验证纳米抗体在抗原表位展示中的作用。方法采用基因合成的方法,将β-HCGC端表位(氨基序列151~165)插入到纳米抗体CDR3区,酶切、连接原核表达载体pET24a,IPTG诱导表达。将His标签填料纯化得到的高纯度目的蛋白免疫新西兰大耳白兔,经5次免疫后,间接ELISA检测效价,抗原偶联纯化介质进行免疫多抗纯化,Western blot进行多抗特异性检测。结果成功构建原核表达重组载体,并获得高表达菌株,IPTG诱导后,目的蛋白主要以包涵体的形式存在,亲和层析获得纯度大于98%的目的蛋白,包涵体经复性后免疫,效价可达1∶512000,Western blot特异性检测显示免疫多抗能够特异性结合β-HCG。结论纳米抗体CDR3区β-HCG抗原C端表位展示的方法,可用于抗β-HCG抗体的制备,并为今后纳米抗体表位展示相关研究奠定基础。Objective To verify the role of nanobodies in epitope display by displaying the C-terminal epitope of β- HCG in the CDR3 region of nanobodies. Methods The C-terminal epitope of β-HCG (amino sequence 151-165) was inserted into the CDR3 region of the nanobody by gene synthesis method. The prokaryotic expression vector pET24a was constructed by digestion and ligation, and the expression was induced by IPTG. His tag filler was purified. The purified target protein was immunized to New Zealand white rabbits. After five immunizations, the indirect ELISA titer was detected and the polyclonal antibody was purified by antigen-coupled purification medium. Western blot assay was used to detect the specificity of polyclonal antibodies. Results Prokaryotic expression vector was constructed, and the highly expressed strains were obtained. After induction by IPTG, the target protein existed mainly in the form of inclusion bodies. Affinity chromatography obtained the target protein with purity greater than 98%. After refolding, the inclusion bodies were immunized with titer of 1∶512 000. Western blot specific detection showed that the immune polyclonal antibody could specifically bind to β-HCG. Conclusion The method of displaying the C-terminal epitope of β-HCG antigen in CDR3 region of nanobody can be used for the preparation of β-HCG antibody, and which lay a foundation for the future research of displaying the epitope of nanobody.

关 键 词：抗体 绒毛膜促性腺激素 表位 纳米抗体 抗体制备 

分 类 号：R392[医药卫生—免疫学]