从大容量噬菌体抗体库获取人源性抗角蛋白抗体ScFv及Diabody的构建

Screening of Human Anti-Keratin ScFv from A Large Phage Antibody Library and Construction of Diabody

目的从大容量噬菌体抗体库筛选人源性抗角蛋白抗体,并构建双体抗体(Diabody)。方法以固相化的角蛋白对构建的大容量噬菌体抗体库进行筛选,经3～4轮筛选后,挑取克隆,ELISA法鉴定其特异性,并对部分抗角蛋白阳性抗体克隆基因进行DNA序列分析。选取活性好的克隆基因进行改造,构建Diabody表达载体。结果在抗体库的筛选过程中可见明显的富集现象,获得了29株可与角蛋白特异性结合的人源单链抗体,选取4个克隆基因进行序列分析,结果表明,4个克隆的轻链基因均属于λ轻链第1亚群,1号和8号克隆的重链基因属于人IgG第2亚群,6号和29号克隆分别属于第1和第3亚群。从表达抗角蛋白抗体的集落中挑选一个进行基因改造,构建的Diabody表达载体所表达的Diabody活性较高。结论利用噬菌体抗体技术获得了人源性抗角蛋白抗体,并改造成应用前景较好的Diabody,为开发银屑病治疗性抗体奠定了基础。

Objective To screen human anti-keratin ScFv from a large phage antibody library and construct diabody.Methods A constructed human phage antibody library was panned against immobilized keratin for 4 times,and the screened clones after 3 ～ 4 rounds of panning were identified for specificity by ELISA.The positive clones against keratin were analyzed by DNA sequencing.A clone with high affinity to keratin was selected to construct a diabody expression vector by gene modification.Results Significant enrichment was observed during panning of phage antibody library.After 4 rounds of panning,29 clones specific to keratin were screened,from which 4 were analyzed by gene sequencing.The result showed that all the light chain genes of the 4 clones were subgroup 1 of λ light chain,however,the heavy chain genes of clones 1 and 8 were subgroup 2 of human IgG,and those of clones 6 and 29 were subgroups 1 and 3 respectively.The diabody with high activity was expressed by using the expression vector.Conclusion Human anti-keratin ScFv was obtained by phage antibody technique and prepared into diabody with good prospect,which laid a foundation of developing therapeutic antibody against psoriasis.