摘要
目的:从大容量天然噬菌体抗体库中筛选抗FKBP52人源单链抗体,并进一步构建其真核表达载体。方法:以FKBP52为抗原,通过吸附、扩增、洗脱等过程,从大容量天然噬菌体抗体库中筛选特异性单链抗体,采用ELISA方法进行特异性测定,采用生物膜干涉方法测定亲和力,再经同源重组方法构建完整抗体的真核表达载体,通过PCR及序列测定对构建的载体进行验证。结果:经过4轮筛选,获得15种与FKBP52特异结合的噬菌体抗体,其中7种得到可溶性表达;序列分析表明,7种单链抗体重链可变区分属VHⅠ、VHⅢ和VHⅣ亚群,轻链可变区分属VκⅠ、VκⅢ和VλⅠ亚群;特异性结合活性较好的4株抗体的亲和常数分别为9.723×10-8、3.500×10-8、1.203×10-8和5.323×10-8;将亲和力较好的一株单链抗体轻、重链可变区基因分别拼接到含有人κ及Ig G1恒定区基因的p CMV-L和p CMV-H真核表达载体中。结论:筛选获得多株人源抗FKBP52单链抗体,并构建了完整抗体的真核表达载体。
Objective：To clone scFvs against human FKBP52 from a large phage antibody library and to construct eukaryotic expression vectors.Methods：FKBP52 was used as the antigen to screen specific scFvs from a large phage antibody library by adsorption,amplification and elution.The specificity and affinity of the scFvs were determined by ELISA and Bio-layer interferometry respectively.The eukaryotic expression vectors were constructed by homologous recombination and verified by PCR and sequencing.Results：Fifteen specific kinds of phage antibodies against FKBP52 were obtained after four rounds of screening,seven of which were successfully expressed as soluble scFvs.The VH regions of the seven soluble scFvs belong to VH Ⅰ,VH Ⅲ and VHⅣ subgroups and the VL regions belong to Vk Ⅰ,VκⅢ and Vλ Ⅰ subgroups.The affinity of four specific scFvs were determined,their affinity constants（K_D） were 9.723×10^（-8）,3.500×10^（-8）,1.203×10^（-8） and 5.323×10^（-8） respectively.One of the four scFvs which had a better affinity was chosen to successfully assemble an eukaryotic expression vector which expresses whole human IgG.Conclusion：Specific scFvs against human FKBP52 were obtained from a large phage antibody library and an eukaryotic expression vector of complete human IgG was also successfully constructed.
出处
《生物技术通讯》
CAS
2015年第6期786-789,共4页
Letters in Biotechnology
基金
国家自然科学基金(81273311
81172119)
