【摘要】 目的:探讨G145R rHBsAg抗原衰减对抗体亲和纯化的影响与意义。方法:采用抗野生重组HBs G6-McAb制备层析载体,对含rG145R HBsAg的2A8细胞上清做亲和层析。以SDS-PAGE、Western Blot及ELISA等对产物纯度、含量及回收率进行评价,并与同法纯化之野生HBsAg进行比较。结果:梯度洗脱层析显示G145R rHBsAg、自然表达野生HBsAg及其r-wHBsAg三者纯化产物的纯度分别为90.3%、95.2%及93.1%;回收率为43.3%、72.0%及66.4%,其亲和洗脱峰型前者较后两者略宽,主峰前部出现明显顿挫;pH线性梯度洗脱显示,G145R rHBsAg洗脱曲线主峰较前梯度洗脱进一步增宽,顿挫更为明显,并在主峰前出现一低平的蛋白峰。ELISA检测显示HBsAg活性贯穿主峰始终,SDS-PAGE与Weistern blot显示两法洗脱产物纯度(92.5%与89.3%)大致相似,分子大小与野生HBsAg相同。结论:洗脱峰加宽与顿挫作为G145RrHBsAg抗原性渐进性衰减的特征性表现,在同类生物材料实验性制备与产品考评中有一定参考价值。更多还原

【Abstract】 Objective:To observe the influence of G145R Mutation of Recombinant Hepatitis B surface antigen attenuation to antibody affinity chromatography purification.Method:The culture supernatant of 2A8 mutant cell line expressing G145R rHBsAg was affinitied by G6-McAb.SDS-PAGE,Western Blot,and several different ELISA identified the purity,the quantity and the recovery rate of the purification.The same ways were used in the samples of HBV patients’serum,wide type recombinant HBsAg.Result:The purity of G145R rHBsAg,HBV patients’serum,and wide type recombinant HBsAg were 90.3%,95.2% and 93.1% respectively;The recovery rate was 43.3%,72% and 66.4% respectively.The pictures of the purifications showed that the peak of G145R rHBsAg was wider than that of the other two,obvious pause and transition in melody before the main peak;PH linearity grads showed that the G145R rHBsAg main peak was more wider than previous grads and there were a low protein peak before the main peak.ELISA showed that HBsAg activation capability exist in the main peak.SDS-PAGE and Weistern blot showed that the purity of two parts was similar(92.5% and 89.3 %).Conclusion:The widen and the conspicuous tortuosity of the main eluting peak were the feature of antigen gradual attenuation,there were certain reference worth in the biomaterial experimental preparation and the products’ check and estimate.更多还原