无血清悬浮培养PK-15细胞及其对猪圆环病毒2型增殖的影响

doi:10.16431/j.cnki.1671-7236.2022.06.039

摘要/Abstract

摘要： 【目的】建立基于无血清悬浮培养PK-15细胞生产猪圆环病毒2型（PCV2）疫苗的工艺，提高PCV2疫苗生产效率，降低PCV2疫苗生产成本。【方法】首先采用直接驯化法对贴壁生长的PK-15细胞进行无血清悬浮培养驯化，并在无血清悬浮培养体系下，采用连续传代的方法考察驯化成功的PK-15细胞的传代和生长稳定性。研究不同感染复数（MOI）（0.10、0.05、0.01和0.001）和不同PK-15细胞接种密度（CDI）（1.0×10⁶、3.0×10⁶、5.0×10⁶/mL）对PCV2增殖的影响，同时对感染病毒前后的细胞培养液中葡萄糖、氨基酸及代谢副产物乳酸和氨进行初步分析。【结果】贴壁PK-15细胞经过30 d的直接驯化可以快速适应无血清悬浮培养，且驯化过程中细胞平均比生长速率由0.1 d^-1增加到0.6 d^-1；悬浮PK-15细胞可以至少连续稳定传15代，连续传代过程中平均比生长速率在0.6 d^-1附近波动，且细胞活率始终>90%；以1.0×10⁶/mL接种，第4天可达到峰值活细胞密度6.2×10⁶/mL，并可维持1 d，第4天前活率均>90%，此后快速下降；病毒增殖最佳工艺参数为：感染复数为0.05，细胞接种密度为1.0×10⁶/mL，最终收获时病毒滴度可达10^6.2TCID₅₀/mL；对细胞感染前后的代谢分析发现，病毒感染后细胞对葡萄糖和多数氨基酸代谢快于感染前，且感染组在感染后72 h附近出现葡萄糖和谷氨酰胺耗竭并伴随代谢副产物乳酸和氨快速积累，之后细胞改变代谢途径并利用乳酸。【结论】 30 d的直接驯化可以获得悬浮PK-15细胞株，PK-15细胞可用于PCV2增殖，结果可为大规模无血清悬浮培养PK-15细胞生产PCV2疫苗提供一定理论和实践基础。

关键词: PK-15细胞; 无血清悬浮培养; 猪圆环病毒2型(PCV2); 增殖; 氨基酸代谢

Abstract: 【Objective】 The aim of this study was to develop a process for producing Porcine circovirus type 2(PCV2) vaccine based on serum-free suspension culture of PK-15 cells, improve the production efficiency, and reduce the production cost.【Method】 Adherent PK-15 cells were domesticated as suspension cells in serum-free medium by direct domestication method.Then, in serum-free suspension culture system, the domesticated PK-15 cells were continuously passaged to investigate the passage and growth stability.Further, the process parameter such as multiplicity of infection (MOI) (0.10, 0.05, 0.01 and 0.001) and cell density at infection (CDI) (1×10⁶, 3×10⁶ and 5×10⁶/mL) was explored in a 125 mL shake flask.At the same time, the difference of glucose, amino acid, and metabolic byproducts such as lacitc acid, ammonium metabolism in the culture medium before and after cell infection with virus was studied.【Result】 After 30 days direct domestication, PK-15 cells could completely adapt to serum-free suspension culture.The average specific cell growth rate was increased from 0.1 d^-1 to 0.6 d^-1 during domestication.Suspension PK-15 cells could be continuously and stably passaged 15 times, and the average specific growth rate was about 0.6 d^-1, and the cell viability was always>90%.At inoculation density 1.0×10⁶/mL, the peak viable cell density reached 6.2×10⁶/mL on the 4th day and maintained for 1 day.The viability before the 4th day was more than 90%, and then rapidly declined.In the process of virus proliferation, the optimal process parameters for virus production were determined:MOI=0.05, CDI=1.0×10⁶/mL.The virus titer at the final harvest reached 10^6.2 TCID₅₀/mL.Metabolic analysis of cells before and after infection showed that the metabolic rates of glucose, most amino acid were significantly accelerated after virus infection.In the infected group, glucose and glutamine depletion occurred around 72 h, accompanied by rapid accumulation of metabolic by-products lacitc acid and ammonia, then cells would change the metabolic pathway to use lacitc acid.【Conclusion】 Suspended PK-15 cells could be obtained after 30 days direct domestication, and suspended PK-15 cells could be used for proliferation of PCV2.The results provided a theoretical and practical basis for large-scale serum-free suspension culture of PK-15 cells to produce PCV2 vaccine.

Key words: PK-15 cells; serum-free suspension culture; Porcine circovirus type 2 (PCV2); proliferation; amino acid metabolism

中图分类号:

S852.65⁺1

周伟, 刘旭平, 张艳敏, 彭雯娟, 赵亮, 谭文松. 无血清悬浮培养PK-15细胞及其对猪圆环病毒2型增殖的影响[J]. 中国畜牧兽医, 2022, 49(6): 2397-2405.

ZHOU Wei, LIU Xuping, ZHANG Yanmin, PENG Wenjuan, ZHAO Liang, TAN Wensong. Serum-free Suspension Culture of PK-15 Cells and Its Effect on the Proliferation of Porcine Circovirus Type 2[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2397-2405.

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