Establishment of MDCK Stable Cell Lines Expressing TMPRSS2 and MSPL and Their Applications in Propagating Influenza Vaccine Viruses in Absence of Exogenous Trypsin.

Biotechnology Research International Pub Date : 2015-01-01 Epub Date: 2015-03-30 DOI:10.1155/2015/402628
Zhiyuan Wen, Chao Wu, Weiye Chen, Xianying Zeng, Jianzhong Shi, Jinying Ge, Hualan Chen, Zhigao Bu
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引用次数: 8

Abstract

We established two Madin-Darby canine kidney (MDCK) cell lines stably expressing human airway transmembrane protease: transmembrane protease, serine 2 (TMPRSS2) and mosaic serine protease large form (MSPL) which support multicycle growth of two H5 highly pathogenic avian influenza viruses (HPAIV) recombinant vaccines (Re-5 and Re-6) and an H9 avian influenza virus (AIV) recombinant vaccine (Re-9) in the absence of trypsin. Data showed that the cell lines stably expressed TMPRSS2 and MSPL after 20 serial passages. Both MDCK-TMPRSS2 and MDCK-MSPL could proteolytically cleave the HA of Re-5, Re-6, and Re-9 and supported high-titer growth of the vaccine without exogenous trypsin. Re-5, Re-6, and Re-9 efficiently infected and replicated within MDCK-TMPRSS2 and MDCK-MSPL cells and viral titer were comparable to the virus grown in MDCK cells with TPCK-trypsin. Thus, our results indicate a potential application for these cell lines in cell-based influenza vaccine production and may serve as a useful tool for HA proteolytic cleavage-related studies.

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表达TMPRSS2和MSPL的MDCK稳定细胞系的建立及其在缺乏外源性胰蛋白酶的流感疫苗病毒繁殖中的应用
建立了两株稳定表达人气道跨膜蛋白酶的Madin-Darby犬肾(MDCK)细胞株:跨膜蛋白酶丝氨酸2 (TMPRSS2)和大形式花叶丝氨酸蛋白酶(MSPL),它们支持H5高致病性禽流感病毒(HPAIV)重组疫苗(Re-5和Re-6)和H9禽流感病毒(AIV)重组疫苗(Re-9)在缺乏胰蛋白酶的情况下的多周期生长。数据显示,经过20次连续传代,细胞系稳定表达TMPRSS2和MSPL。MDCK-TMPRSS2和MDCK-MSPL均能蛋白裂解Re-5、Re-6和Re-9的HA,支持疫苗在没有外源性胰蛋白酶的情况下高滴度生长。Re-5、Re-6和Re-9在MDCK- tmprss2和MDCK- mspl细胞中有效感染和复制,病毒滴度与在MDCK细胞中使用TPCK-trypsin生长的病毒相当。因此,我们的研究结果表明这些细胞系在基于细胞的流感疫苗生产中的潜在应用,并可能作为HA蛋白水解裂解相关研究的有用工具。
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