张洪波 赵成跟 黄兴建

摘要：目的 研究降纤酶制备工艺中β-丙内酯对脂包膜指示病毒的灭活能力，为该产品病毒安全性评价提供依据。方法 用微量滴定法测定β-丙内酯工艺处理前后各批次样品中指示病毒VACV和VSV的滴度，按Reed-Muench法計算病毒滴度，并计算β-丙内酯处理前后各批次样品中各指示病毒的滴度下降值。结果 β-丙内酯静置灭活2h后，蛇毒溶液中VACV滴度均降至检出限1.7 logs以下，滴度平均下降值≥4.3 logs;8h后，蛇毒溶液中VSV滴度均降至检出限1.7 logs以下，滴度平均下降值≥5.0 logs。对照组蛇毒溶液静置24h后VACV滴度平均下降0.4 logs，VSV滴度平均下降0.3 logs。结论 降纤酶制备工艺中的β-丙内酯工艺是一个有效的灭活脂包膜病毒的工艺步骤。

关键词：尖吻蝮蛇蛇毒;β-丙内酯;VACV;VSV

Abstract： Objective To study the ability of β-Propiolactone to inactivate lipid-encapsulated indicator viruses in the preparation process of defibrase， and to provide a basis for the virus safety evaluation of the product. Methods Microtiter method was used to determine the titers of indicator viruses VACV and VSV in each batch of samples before and after the β-Propiolactone process. The virus titer was calculated according to the Reed-Muench method， and the titer of each indicator virus in each batch of samples before and after the β-Propiolactone treatment was calculated. Decline value. Results After the BPL was inactivated for 2 hours， the VACV titer in the snake venom solution fell below the detection limit of 1.7 logs， and the average titer decrease was ≥4.3 logs. After 8 hours， the VSV titer in the snake venom solution fell to the detection limit of 1.7. Below logs， the average drop in titer is ≥5.0 logs. The VACV titer of the snake venom solution in the control group decreased by 0.4 logs and the VSV titer decreased by 0.3 logs after being allowed to stand for 24 hours. Conclusion The β-Propiolactone process in the preparation of defibrase is an effective process step for inactivating lipid-enveloped viruses.

Keywords： Agkistrodon  venom; BPL; VACV; VSV

【中图分类号】R97             【文献标识码】A             【文章编号】2107-2306（2021）09--03

降纤酶是从尖吻蝮蛇蛇毒中提取分离得到的一种类凝血酶，是以丝氨酸为活性中心的蛋白水解酶，直接作用于纤维蛋白原，释放血纤肽A（FPA），生成非交联的纤维蛋白，具有显著的去纤、降粘、溶栓等作用，临床上广泛应用于治疗和预防心脑血管血栓性疾病[1]。

然而蛇毒粗毒成分非常复杂， 除类凝血酶外还含有神经毒素、细胞毒素、肌肉毒素等多种活性成分，为引起各种毒副作用的主要原因[2];现行降纤酶质量标准要求降纤酶产品纯度达到SDS-PAGE电泳纯， HPLC测定纯度需大于90%[3]，而天然蛇毒中的类凝血酶绝大多数为酸性蛋白质， 经典的蛋白质提纯方法如盐析、热变性、酸碱变性、有机溶剂沉淀、超速离心等， 由于专一性差和分辨率不高，不能有效去除病毒类杂质，已很少用于降纤酶的制备。……