大疱性类天疱疮患者噬菌体抗体库的构建及抗BP180-NC16A抗体筛选

Construction of Phage Antibody Library and Isolation of Anti-BP180-NC16A Antibodies from Patient with Bullous Pemphigoid

目的构建噬菌体抗体库并筛选抗BP180-NC16A抗体。方法以大疱性类天疱疮(BP)患者外周血淋巴细胞为基因来源,扩增多样性的轻链和重链Fd段基因,构建Fab段表面展示的噬菌体抗体库,用BP180分子的NC16A片段为抗原对抗体库进行筛选并对筛选得到的抗体用ELISA,W estern b lot和免疫荧光等方法进行鉴定。结果经过轻、重链基因的重组和转化,获得了库容为5×107的噬菌体抗体库。以BP180-NC16A抗原进行"亲和吸附—洗脱—扩增"淘筛,获得2株特异性抗体。结论利用噬菌体抗体库技术成功制备了抗BP180抗体,为深入研究BP自身抗体、探讨新的治疗策略奠定了基础。

Objective To produce anti-BP180-NC16A Fab antibody fragments from the antibody repertiore of the patient with bullous pemphigoid （BP）. Methods Phage antibody library was constructed with diversified IgG light chain genes and Fd fragments amplified from the lymphocytes isolated from a BP patient. Panning of the library against the recombinant NC16A domain of human BP180 was conducted to select specific antibodies. The binding activity and specificity of the obtained antibodies were determined by ELISA, Western blot and immunofluoreseence. Results A phage antibody library with the capacity of 5 × 10^7 was constructed successfully. Two individual clones of anti-BP180-NC16A antibodies were selected from the library. By characterization, these 2 monoclonal Fab antibodies showed specific binding to human BP180 and linear deposition along dermal-epidermal junction of human skin. Conclusion It is feasible to generate specific anti-BP180-NC16A Fab antibodies using genetically engineering technology. It would be useful in furthering the studies on the pathogenesis and treatment of BP.