重组抗HBsAg单链抗体在HBV转基因小鼠体内作用的研究

In vivo binding activity of recombinant ScFv against HBsAg in HBV transgenic mice

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 investigate the activity of recombinant single-chain Fv against HBsAg (HBscFv) in HBV transgenic mice, and the possibility of applying HBV transgenic mice as a model to evaluate the binding-activity of HBsAg specific antibodies. Methods Produced from recombinant E.coli M15[pQE-HBscFv], inclusion body of HBscFv was purified by immobilized metal chelation and gel filtration chromatography, and then renaturated by dialysis step-by-step. HBscFv product was injected into the tail vein of HBV transgenic mice and the concentration of HBsAg in mice sera was determined by sandwich ELISA. The decreasing degree of HBsAg concentration, defined as binding rate, was calculated. At the same time, HBsAb IgG from human blood was used as positive control, with 0.9% NaCl as negative control. Results The recombinant HBscFv was able to be purified to a purity as high as 98% by two-step method. The binding rate of HBscFv was (30.47±9.85)%, that was different significantly with that of NaCl solution ( P <0.001) and not different with that of HBsAb IgG ( P >0.05). The binding rate of HBsAb IgG was (39.00±7.43)%, that was different significantly with that of NaCl solution ( P <0.001). Comparing the binding rate and protein concentration of HBscFv with those of HBsAb IgG, the binding titer of HBscFv was determined to be 31.58U/mg. Conclusion HBscFv has in vivo HBsAg-binding activity in HBV transgenic mice, which might be developed into a candidate model to evaluate the in vivo binding activity of HBsAg specific antibodies.