Abstract:

To explore the feasibility of using recombinant trypsin as a substitute for porcine derived trypsin in the preparation of attenuated Japanese encephalitis live vaccine， porcine derived trypsin， recombinant trypsin derived from Escherichia coli BL21 （DE3） expression system， and recombinant trypsin derived from Pichia pastoris （X-33） expression system were used to digest primary hamster kidney blocks at the same enzyme activity （total enzyme activity 315 200 U）， with the same digestion temperature and time in each group. The results showed that after digestion with three types of pancreatic enzymes， the cell dispersion effect and growth status were good， and there was no significant difference in the cell spreading rate on the third day of cultivation （P>0.05）. During the virus culture stage， there was no significant difference in virus titers among the three groups of virus supernatants （P>0.05）. The results showed that recombinant trypsin derived from E. coli BL21 （DE3） expression system and Pichia pastoris （X-33） expression system can replace porcine trypsin in the preparation of attenuated live Japanese encephalitis vaccine.

Key words: Japanese encephalitis vaccine, porcine pancreatic enzyme, recombinant trypsin, cellular digestion

摘要：

为探究重组胰蛋白酶替代猪源性胰蛋白酶在乙型脑炎减毒活疫苗制备中的可行性，在相同的酶活力条件（总酶活力均为315 200 U）下，利用猪源性胰蛋白酶、源于大肠杆菌BL21（DE3）表达系统的重组胰蛋白酶，以及源于毕赤酵母（X-33）表达系统的重组胰蛋白酶，分别消化原代地鼠肾块，各组实验均保持相同的消化温度和消化时间。结果发现，经上述3种胰酶消化后，细胞的分散效果及细胞生长状态均表现良好，且在培养第3天的细胞铺展率上，各组间并未显示出明显差异（P>0.05）。进一步在病毒培养阶段观察，3个组别病毒上清液的病毒滴度亦无明显差异（P>0.05）。结果表明，源于大肠杆菌BL21（DE3）表达系统和毕赤酵母（X-33）表达系统的重组胰蛋白酶，均可替代猪源性胰蛋白酶，用于乙型脑炎减毒活疫苗的制备。

关键词: 乙脑疫苗, 猪源胰酶, 重组胰蛋白酶, 细胞消化