發(fā)布時(shí)間：2018-07-06 10:16

  本文選題：PK-15細(xì)胞 + 低血清培養(yǎng)基��； 參考：《華東理工大學(xué)》2015年碩士論文

【摘要】：豬腎細(xì)胞(PK-15)對(duì)多種豬類病毒敏感,廣泛應(yīng)用于獸用疫苗的生產(chǎn)和研發(fā)。本文開發(fā)了適于PK-15細(xì)胞生長的廉價(jià)低血清培養(yǎng)基P-LSM(含3%新生牛血清),該培養(yǎng)基適于PK-15細(xì)胞靜置培養(yǎng)和微載體懸浮培養(yǎng),并且利于豬細(xì)小病毒的繁殖。本文開發(fā)了基于PK-15細(xì)胞低血清靜置培養(yǎng)的豬細(xì)小病毒生產(chǎn)工藝,最大病毒滴度達(dá)到7.5Lg TCID50/ml。成功將此工藝從靜置培養(yǎng)轉(zhuǎn)移到微載體懸浮培養(yǎng),并完成了1.5L和5L反應(yīng)器上的驗(yàn)證實(shí)驗(yàn),最大病毒滴度達(dá)到7.2Lg TCID50/ml。最大病毒滴度較生產(chǎn)企業(yè)增加了10倍。并首次發(fā)現(xiàn)了乳酸對(duì)葡萄糖得率與病毒滴度的正相關(guān)性,可作為指針病毒滴度以及收毒時(shí)間的重要參數(shù)。利用13C標(biāo)記葡萄糖和谷氨酰胺的代謝流分析技術(shù),本文就PK-15細(xì)胞的代謝和豬細(xì)小病毒接種PK-15細(xì)胞前后的細(xì)胞代謝變化進(jìn)行了初步研究,并初步構(gòu)建了PK-15細(xì)胞中心碳代謝的網(wǎng)絡(luò)模型。研究發(fā)現(xiàn)在細(xì)胞延滯期,葡萄糖主要進(jìn)行有氧糖酵解；指數(shù)生長期,部分葡萄糖開始通過丙酮酸脫氫酶經(jīng)乙酰輔酶A進(jìn)入TCA循環(huán)；進(jìn)入穩(wěn)定期后,部分葡萄糖開始通過丙酮酸羧化酶進(jìn)入TCA循環(huán)；谷氨酰胺主要為TCA循環(huán)提供能量和中間代謝物。PK-15細(xì)胞接種病毒后,更多的葡萄糖通過磷酸戊糖途徑、Ser-Gly途徑和天冬氨酸,進(jìn)入核苷酸合成；更多谷氨酰胺轉(zhuǎn)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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S859.797;Q813.1

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本文編號(hào)：2102450