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目的应用短波UVC对人凝血酶原复合物(Prothrombin complex concentrate,PCC)进行灭活处理,验证病毒灭活效果及对产品质量的影响。方法以猪细小病毒(Porcine parvovirus,PPV)作为指示病毒,采用短波UVC对PCC样品进行灭活,紫外辐射剂量分别为200、250和300 J/m2,微量细胞培养法检测病毒滴度,对未出现病变的样品盲传3代,验证灭活效果;应用二维电泳(2-DE)、高效液相色谱技术(HPLC)及活性检测方法对PCC样品的结构和功能及活性进行评价。结果短波UVC法可使PPV滴度降低4.0 log以上,所有样品在盲传第3代时均检测到细胞病变。在无保护剂的情况下,凝血因子活性回收率大于70%;HPLC分析结果证实,UVC照射后,凝血因子蛋白聚合体含量无明显改变,与照射前比较,蛋白的色谱行为基本一致;2-DE检测显示,样品照射前后,图谱蛋白斑点数量基本相等,其中300 J/m2 UVC处理组有2个蛋白点群出现位置和灰度值的变化。结论短波UVC对无胞膜病毒灭活效果较好,且该灭活方法对PCC制品蛋白活性影响较小。
Objective To inactivate human Prothrombin complex concentrate (PCC) with short-wave UVC to verify the effect of virus inactivation and product quality. Methods Porcine parvovirus (Porcine parvovirus) was used as the indicator virus. The PCC samples were inactivated by shortwave UVC. The doses of ultraviolet radiation were 200, 250 and 300 J / m2 respectively. The titer of virus was detected by micro-cell culture method. Pathological samples were blindly passaged for 3 generations to verify the inactivation effect. The structure, function and activity of PCC samples were evaluated by 2-DE, HPLC and activity assay. Results Shortwave UVC method could reduce the titer of PPV by more than 4.0 log, and all the samples detected cytopathic effect on the third generation of blind passage. In the absence of protective agent, the recovery rate of coagulation factor activity was more than 70%. The results of HPLC analysis confirmed that there was no significant change in the coagulation factor protein aggregates after UVC irradiation. The chromatographic behaviors of 2- The results of DE assay showed that the number of spots in the spectra was almost the same before and after the irradiation. There were two protein spots in the 300 J / m2 UVC treatment group, and the spots and gray values changed. Conclusion Short-wave UVC has a good effect on inactivating the mycelium virus, and the inactivation method has little effect on the protein activity of PCC products.