PK-15细胞低血清微载体悬浮培养生产猪细小病毒疫苗的工艺开发和代谢流研究

发布时间：2018-07-06 10:16

本文选题：PK-15细胞 + 低血清培养基　；参考：《华东理工大学》2015年硕士论文

【摘要】：猪肾细胞(PK-15)对多种猪类病毒敏感,广泛应用于兽用疫苗的生产和研发。本文开发了适于PK-15细胞生长的廉价低血清培养基P-LSM(含3%新生牛血清),该培养基适于PK-15细胞静置培养和微载体悬浮培养,并且利于猪细小病毒的繁殖。本文开发了基于PK-15细胞低血清静置培养的猪细小病毒生产工艺,最大病毒滴度达到7.5Lg TCID50/ml。成功将此工艺从静置培养转移到微载体悬浮培养,并完成了1.5L和5L反应器上的验证实验,最大病毒滴度达到7.2Lg TCID50/ml。最大病毒滴度较生产企业增加了10倍。并首次发现了乳酸对葡萄糖得率与病毒滴度的正相关性,可作为指针病毒滴度以及收毒时间的重要参数。利用13C标记葡萄糖和谷氨酰胺的代谢流分析技术,本文就PK-15细胞的代谢和猪细小病毒接种PK-15细胞前后的细胞代谢变化进行了初步研究,并初步构建了PK-15细胞中心碳代谢的网络模型。研究发现在细胞延滞期,葡萄糖主要进行有氧糖酵解；指数生长期,部分葡萄糖开始通过丙酮酸脱氢酶经乙酰辅酶A进入TCA循环；进入稳定期后,部分葡萄糖开始通过丙酮酸羧化酶进入TCA循环；谷氨酰胺主要为TCA循环提供能量和中间代谢物。PK-15细胞接种病毒后,更多的葡萄糖通过磷酸戊糖途径、Ser-Gly途径和天冬氨酸,进入核苷酸合成；更多谷氨酰胺转化成天冬氨酸,合成核苷酸。
[Abstract]:Porcine kidney cells (PK-15) are sensitive to various porcine viruses and are widely used in the production and development of veterinary vaccines. A cheap low serum medium P-LSM (containing 3% newborn bovine serum) was developed for PK-15 cell growth. The medium is suitable for PK-15 cell static culture and microcarrier suspension culture, and is conducive to porcine parvovirus propagation. In this paper, the production process of porcine parvovirus based on PK-15 cell low serum static culture was developed. The maximum virus titer reached 7.5 Lg TCID 50 / ml. The process was successfully transferred from static culture to microcarrier suspension culture, and the verification experiments in 1.5L and 5L reactors were carried out. The maximum virus titer reached 7.2Lg TCID 50 / ml. The maximum virus titer is 10 times higher than that of the manufacturer. The positive correlation between the yield of lactic acid to glucose and the titer of virus was found for the first time, which can be used as an important parameter of virus titer and time of virus collection. The metabolism of PK-15 cells and the metabolic changes of porcine parvovirus before and after inoculation of PK-15 cells were studied using 13C-labeled glucose and glutamine metabolic flow analysis technique. A network model of central carbon metabolism in PK-15 cells was constructed. The study found that during cell delay, glucose is mainly glycolytic; during exponential growth, part of glucose begins to enter TCA cycle through acetyl coenzyme A through pyruvate dehydrogenase; after entering stable phase, Some glucose began to enter the TCA cycle through pyruvate carboxylase, and glutamine mainly supplied the TCA cycle with energy and intermediate metabolites. PK-15 cells inoculated with the virus, more glucose passed through the pentose phosphate pathway to the Ser-Gly pathway and aspartic acid. Enter nucleotide synthesis; more glutamine is converted into aspartic acid to synthesize nucleotides.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.797;Q813.1

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