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