【摘要】 对大肠杆菌表达的人源性抗乙型肝炎表面抗原 (HBsAg)单链抗体 (scFv)进行了纯化研究 ,并对单链抗体活性及其结构进行了鉴定 .大肠杆菌表达的单链抗体包涵体 ,用8mol/L尿素裂解 ,经过Ni离子螯合亲和层析和凝胶过滤两步纯化后 ,纯度 (w)达到97%以上 ,再通过透析复性除去尿素 .经间接ELISA和竞争性ELISA证明 ,复性后的scFv具有与HBsAg结合的活性 ,而且对鼠源抗HBsAg单克隆抗体的抑制率可达 4 0 .3% .基质辅助激光解析飞行时间质谱 (matrixassistedlaserdesorptionionization ,MALDI_TOF_MS)的结果证明 ,重组的scFv分子量与理论值相符 ,肽质量图谱的结果证明重组单链抗体的一级结构与理论序列是完全相符的 ,并确定了scFv蛋白中二硫键的位置更多还原

【Abstract】 The purification of a human anti_HBsAg single chain Fv(scFv) expressed by E. Coli was researched and the bioactivity and structure of the scFv was indentified.The scFv formed inclusion body in E. Coli. 8 mol/L urea was used to solubilize the inclusion body, and the scFv was then purified in two steps , including Ni 2+ chelating affinity chromatography and gel filtration. After purification, the purity of the scFv reached 97%. The results of direct ELISA and competitive ELISA proved that the refolding scFv could be bound to HBsAg. Furthermore, it could inhibit the murine anti_HBsAg monclonal antibody to bind to HBsAg and the inhibit rate reached 40.3%. The MALDI_TOF_MS was applied to measure the molecular weight of scFv. The experiment result was in conformity with the theoretical volume. The peptide mass mapping confirmed that the primary structure of scFv was correct and could determine the position of disulfide bond.更多还原