猪圆环病毒2型在转管微载体培养PK-15细胞中增殖动态

Replication of porcine circovirus type2 in PK-15 cells on micro carrier incubated in a tubular miniature reactor

为提高猪圆环病毒2型（PCV2）的增殖规模及单位体积内的病毒滴度,本研究利用转管微型反应器,采用荧光定量PCR检测方法,对PCV2在PK-15细胞中复制特性和增殖动态进行了系统研究。结果显示,转管（内置0.6 g纸片载体）中接种约2.98×10^7个DF-1细胞,培养7 d细胞增至1.93×10^8～2.0×10^8个,并确定培养7 d时为最佳接毒时间;病毒的最佳接种剂量为120.8 MOI,最佳收毒时间为接毒后的100 h,可达5.53E＋06以上。细胞数增长与糖耗间呈明显平行关系,在细胞生长期内（168 h）平均每个细胞耗糖量为3.09×10^-8g/24 h,可以根据糖耗量的多少推测细胞生长状态和数量。相同培养液中利用转管微型反应器培养PK-15细胞生产PCV2最终获得的病毒液毒价比转瓶培养毒价提高1.65倍。本实验为PCV2在生物反应器中大规模培养提供了参考依据。

In order to improve the dimensions and virus titer in cell culture, the porcine circovirus type 2 （PCV2） was inoculated in PK-15 cells on micro carrier incubated in a tubular miniature reactor, and the PCV2 replication dynamics were monitored by real-time PCR to optimize the cell culture conditions. The results showed that the PK-15 cells inoculated in tubular （built-in 0.6 g in tubular） at about 2.98×10^7 cells multiplied to around 2.0×10^8 after 7 days when it was suitable for PCV2 inoculation with optimal dose of 120.8 MOI and collected at 100 hours post the inoculation. In addition, the number of cell growth and sugar consumption had a parallel correlation which the average sugar consumption of each cell in cell growth period （168 hours） was up to 3.09×10^8 g/24 hours. While, the virus titers on PK-15 cell at micro carrier in tubular miniature reactor were about 1.65 times higher than that of roller bottle. These results provided the parameters for PCV2 replication in PK-15 cell on micro carrier at tubular miniature reactor.