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目的 研究重组人源抗HBsAg单链抗体 (HBscFv)在HBV转基因小鼠体内的活性作用 ,探讨HBV转基因小鼠作为HBsAg特异性抗体的结合活性评价模型的可行性。方法 工程菌表达的HBscFv包涵体经固定化金属螯合层析和分子排阻层析两步纯化后 ,分步透析复性。复性后的HBscFv(0 .9g L)经尾静脉注射HBV转基因小鼠 ,一定时间后测定鼠血清中HBsAg的浓度 ,计算抗体注射前后HBsAg浓度下降的百分比 (结合率 ) ,同时以人血源HBsAbIgG(40U ml)和生理盐水作为对照。结果 两步纯化获得纯度达到 98%的重组HBscFv。HBscFv制品能够结合转基因小鼠体内的HBsAg,其结合率为 (30 .4 7± 9.85 ) % ,与生理盐水组差异有显著性 (P <0 .0 0 1) ,与HBsAbIgG组差异无显著性(P >0 .0 5 ) ;对照品HBsAbIgG的结合率为 (39.0 0± 7.4 3) % ,与生理盐水组差异也有显著性 (P <0 .0 0 1)。比较HBscFv和HBsAbIgG的结合率及其浓度 ,求得HBscFv的结合效价为 31.5 8U mg。结论 HBscFv在转基因小鼠体内具有特异性结合HBsAg活性 ,HBV转基因小鼠可发展为评价HBsAg特异性抗体的体内结合活性的候选模型。
Objective To study the activity of recombinant human anti-HBsAg single-chain antibody (HBscFv) in HBV transgenic mice and to evaluate the feasibility of using HBsAg-specific antibody in HBV transgenic mice. Methods The HBscFv inclusion bodies expressed by engineered bacteria were purified by two steps of immobilized metal chelate chromatography and size exclusion chromatography. Refolded HBscFv (0.9gL) was injected into the tail vein of HBV transgenic mice. After a certain period of time, the concentration of HBsAg in the serum was measured and the percentage of HBsAg decreased (the binding rate) before and after antibody injection was calculated. At the same time, HBsAbIgG (40U ml) and saline as control. Results Two steps of purification yielded a recombinant HBscFv with a purity of 98%. The HBsFv product could bind to HBsAg in transgenic mice with a binding rate of (30.47 ± 9.85)%, which was significantly different from that of the saline group (P <0.01), but not with the HBsAbIgG group (P> 0.05). The binding rate of reference substance HBsAbIgG was (39.0 ± 7.43)%, which was also significantly different from that of saline group (P <0.01). The binding rate and concentration of HBscFv and HBsAbIgG were compared to find that the binding potency of HBscFv was 31.5 8U mg. Conclusion HBscFv has a specific binding activity to HBsAg in transgenic mice and HBV transgenic mice can be developed as a candidate model for evaluating the in vivo binding activity of HBsAg-specific antibodies.