噬菌体展示技术构建寻常型天疱疮患者桥粒芯糖蛋白3抗体文库

Construction of Antibody Phage Library in Patients With Pemphigus Vulgaris

目的用噬菌体展示技术构建寻常型天疱疮患者桥粒芯糖蛋白 3抗体文库。方法收集寻常型天疱疮患者外周血，分离淋巴细胞抽提 RNA，采用 RT-PCR反应以人的抗体特异性 HuVHBACK、 HuJHFOR和 HuVkBACK、 HuJkFOR引物扩增抗体重链可变区（ VH）和轻链可变区（ VL）基因片段，并将其与接头拼接成 ScFv片段克隆于大肠杆菌。结果抗体基因 ScFv片段与噬菌体基因Ⅲ的产物以融合蛋白的形式在噬菌体表面表达，构建了天疱疮患者的重组噬菌体抗体库， ELISA检测证明 ScFv基因产物具有抗体活性。结论天疱疮噬菌体抗体库的构建为天疱疮发病机理、诊断和治疗研究打下了基础。

Objective To construct antibody library in patient with pemphigus vulgaris using phage display antibody technique. Methods Total RNA was extracted from B cells of patients with pemphigus vulgaris(PV) using polymerase chain reaction, immunoglobulin VH and VL genes were amplified by specific primer of HuVHBACK, HuJHFOR, HuVkBACK and HuJkFOR. The VH and VL genes were then cloned into a phage vector and expressed on the phage surface as a fusion protein with product of geneⅢ . Results The antibody phage library of single chain antibodies of patients with PV was constructed. Conclusion We have obtained EC1-2 and EC3-4 purified protein of Dsg3 from constructed recombinant plasma in our laboratory which will facilitate the study on Dsg3 antibodies.