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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 antibodies from large-capacity phage antibody library and construct diabody. Methods The constructed phage antibody library was screened with immobilized keratin. After 3 to 4 rounds of screening, the clones were picked and characterized by ELISA. The DNA fragments of some anti-keratin-positive antibody clones Sequence analysis. The cloned gene with good activity was selected for transformation and the Diabody expression vector was constructed. Results In the screening process of antibody library, obvious enrichment phenomenon was observed, and 29 strains of human single chain antibodies specific for keratin were obtained. Four cloned genes were selected for sequence analysis. The results showed that the light intensity of four clones The heavy chain genes of the 1st and the 8th clones belonged to the 2nd subgroup of human IgG, while the 6th and 29th subclones belonged to the 1st and the 3rd subgroup respectively. One of the colonies expressing the anti-keratin antibodies was genetically modified to construct the Diabody expression vector which expressed higher activity of Diabody. Conclusion The use of phage antibody technology to obtain human anti-keratin antibodies, and transformed into Diabody with better prospects for the development of therapeutic antibodies for psoriasis laid the foundation.