孙德君等

摘 要：目的：探索H5N1亚型禽流感病毒在MDCK中增殖规律，确定最佳增殖条件。方法 将H5N1亚型禽流感病毒接种到6孔板培养的MDCK细胞进行增殖试验，检测不同病毒感染量、不同浓度TPCK-胰酶，接毒后不同时间病毒的HA滴度。根据确定的最佳增殖条件将病毒接种到微载体培养的MDCK细胞中进行大规模增殖。结果：最佳病毒增殖条件接毒量MOI为5×10-4、TPCK-胰酶浓度为4 μg/mL，在5 L 生物反应器中重复验证，获得稳定的试验结果，病毒血凝价最高为8 log2。结论：本研究为禽流感疫苗的生物反应器规模化生产奠定了基础。

关键词：H5N1亚型禽流感病毒；MDCK细胞；规模化培养

Abstract： To explore the regularity for the multiplication of avian influenza virus subtype H5N1 in MDCK cell culture system and determine the optimal proliferation conditions. H5N1 subtype of avian influenza virus was inoculated into the MDCK cell growing on 6 well plate， and the HA titers of virus at different time were detected in the conditions of different infectious doses， and different concentrations of TPCK-trypsin. The optimal conditions were determined. Then the H5N1 subtype avian influenza virus was grown in microcarrier-based MDCK cell. It was demonstrated that high virus yield with a hemagglutination unit of 8 log2（1∶256） could be obtained under the optimal conditions of multiplication. The result indicated the H5N1 subtype avian influenza virus could be produced in microcarrier-based MDCK cell in a large-scale culture system with a high virus yield and demonstrates the feasibility of the development of mammalian cell-based in influenza vaccine in microcarrier culture systems.

Keywords： H5N1 subtype avian influenza；Madin-Darbin canine kidney （MDCK） cell； large-scale culture

从首次发现禽流感病毒至今，世界上很多国家都大规模爆发过禽流感疫情，给养禽业带来巨大损失。我国是禽流感病毒高爆发区，农业部要求对鸡、鸭、鹅等家禽实施强制免疫，这就需要大量高效、安全、低成本的禽流感疫苗。目前市场上大部分疫苗仍为鸡胚制备，这种疫苗生产需要消耗大量的SPF鸡胚，且劳动强度大、生产周期长、产量不稳定，不利于应对大规模的禽流感疫情爆发。与传统鸡胚生产方法相比，动物细胞培养生产禽流感方法具有很多优势，如抗原匹配性好，可以进行大规模生产和缩短生产周期，自动化控制，减少过敏反应等。研究证明，禽流感病毒在犬肾传代细胞（MDCK）上具有良好的适应性，用其培养禽流感病毒获得的病毒滴度较高。实验中对H5N1亚型禽流感病毒在MDCK细胞中的增殖条件进行探索，以期筛选规模化培养流感病毒的最适条件，为建立新型流感疫苗的生产基质奠定基础。……