摘要
为了分析单链抗体的连接肽长度对其与小分子半抗原结合力的影响,本研究以含(G_4S)_3连接肽的伏马菌素特异单链抗体基因H2为模板,PCR扩增获得含G_4S和(G_4S)_2连接肽的单链抗体编码基因,分别命名为H2-5和H2-10,通过基因工程操作构建到p HENHi载体中,转化大肠杆菌XL1-Blue进行原核表达,纯化后利用ELISA检测单链抗体的亲和力。结果显示含不同长度连接肽的单链抗体均能在大肠杆菌中可溶性表达,单链抗体结合力与连接肽长度的关系为(G_4S)_3>G_4S>(G_4S)_2,即单链抗体H2与其抗原伏马菌素的亲和力最高,其次为H2-5和H2-10抗体。因此,含有较长连接肽的单链抗体能够自身折叠形成有活性的蛋白,并表现出与小分子半抗原更强的结合能力。
To analyze the effect of the linker peptide length of single-chain variable fragment （scFv） antibody on its binding capacity to small molecular antigen, the gene of fumonisin-specific scFv antibody H2 was taken as the template to individually amplify the coding genes of scFv antibodies that containing G4S and （G4S）2 linker, named as H2-5 and H2-10. Then, they were cloned into pHENHi vector and transformed into Escherichia coli XL1-Blue by genetic manipulation. These scFv antibodies were expressed and purified, and their antigen-binding capacities were determined by enzyme-linked immunosorbent assay （ELISA）. The results showed that all scFv antibodies co- ntaining different length of peptide linker can be expressed in soluble form in bacteria. The correlation of the binding capacity of scFv antibody and the linker length is （G4S）3〉G4S〉（G4S）2, which indicated that the H2 antibody had the highest affinity against the fumonisin antigen, followed by the H2-5 and H2-10 antibody. Thus, the scFv antibody with longer linker could form bioactive protein by itself folding and display better binding capacity for small molecular hapten.
作者
夏雪
石玉玲
喻艳琴
冯敏
王婷婷
方蓉
黄顺莉
曾柱
胡祖权
Xia Xue Shi Yuling Yu Yanqin Feng Min Wang Tingting Fang Rong Huang Shunli Zeng Zhu Hu Zuquan(Key Laboratory of Cell and Gene Engineering, Guiyang, 55000)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2016年第9期2429-2434,共6页
Genomics and Applied Biology
基金
国家自然科学基金(31260227
31660258)
中国博士后科学基金(2015M582758XB)
贵州省科技创新人才团队项目(黔科合人才团队(2015)4021)
贵州省科技项目(黔科合J字[2013]2058号和黔科合LH字[2014]7092)
贵州省高校优秀科技创新人才支持计划(黔教合KY字[2014]244)
贵阳市科技计划项目(筑科合同[2014]37号)
国家级大学生创新训练计划(201310660004)共同资助
关键词
单链抗体
连接肽
小分子半抗原
结合力
Single-chain variable fragment （scFv）, Linker, Small molecular hapten, Binding capacity
