초록
부착의존성 세포주인 Trichoplusia ni 의 유래의 BTI-TN5B1-4 (TN5) 곤충세포주를 이용하여 인간 혈소판생성축진인자인 재조합 인간 트롬보포이에틴(rhTPO)의 배양조건 최적화 연구를 수행하였다. 배양배지, 세포감염에 투입되는 재조합 베큘로바이러스와 숙주세포의 비율(MOI),세포감염시 세포밀도, 배지 회수시간 및 배양방법 등이 rhTPO 의 생산에 미치는 효과를 연구하여 60 mm dish로 정체 배양시 10 MOl 이상,$2\times10^6$ cells 의 세포밀도,바이러스 감염 후72 시간에서 rhTP0 의 최대 발현양 (약 12 mg/L)을 나타내었다. 배양 배지로서는 EXCELL FIVE 배지가 SF900II나Insect serum free media-1 Figure 5. Effect of growth phases on rhTPO production. TN5 cells were grown as suspension culture in 1 L spinner flask with 200 mL of SF900II serum free medium at 80 rpm. The cells were infected with AcBac404-2 at MOl of 1. Culture medium was collected at given time intervals and the expression level of rhTPO was analyzed by ELISA (A) or immunoblot analysis (B). Lanes 1 and 7; cell density of $0.6\times10^6$ cells/mL, lanes 2 and 8; cell density of $1.6\times10^6$ cells/mL, lanes 3 and 9; cell density of $2.0\times10^6$ cells/mL, lanes 4 and 10; cell density of $3.0\times10^6$ cells/mL, lane M; prestained molecular weight marker (Bio-Rad). Lanes 1, 2, 3, and 4; culture medium was collected at 48 hpi and lanes 7, 8, 9, and 10; culture medium was collected at 72 hpi. Figure 6. Effect of culture media on rhTPO production. TN5 cells grown with different culture media were infected with AcBac-404-2 at 10 MOL 10$\mu$L of culture medium was run on SDS-PAGE and Immunoblot analysis was performed. Lane ];TN5 cells cultured with SF900II serum free media(Gibco),and lane 3; TN5 cells cultured with EXPRESS FIVE serum free media (Gibco) 에 비해 더 증가된 발현양을 나타내었다. TN5 세포주를 0.2 L 규모 (1 L spinner flask)oJl에서 세포간의 응집현상 없이 부유배양에 적응,배양시킨 후 세포성장 시기에 따른 발현을 조사한 결과 1 MOI의 감염조건 하에서는 $0.6\times10^6$cell/mL의 early exponential시기의 세포밀도에서 72시간 배양하였을 대 최대 발현양을 나타내었다. 나타내었다.
In order to obtain high-level production of recombinant human thrombopoietin (rhTPO) in insect cell line, HTI-TN-5B1-4 (TN5), conditions for optimal rhTPO expression such as multiplicity of infection (MOI), the cell density at infection, harvesting time and type of culture method as well as growth media were determined. When TN5 cells were cultured as anchorage-dependent state in 60-mm dish, cell density $2\times^6$ cells,MOI of 10 and Garvesting the culture media at 72 hr post-infection wrere the cinditions for highest rh TPO production. High production of rhTPO was also achieved by using EXPRESS FIVE serum free media rather than SF900II serum free media-1. Anchorage-dependent TN5 cells were adapted as a suspension culture when they were grown in the presence of heparin. TN5 cells were successfully cultured at 0.2 L scale in suspension culture without having aggregation. When TN5 cells were cultured as suspension state, cell density of $0.6\times10^6$ cells/mL, MOI of 1 and harvesting the culture media at 72 hr post-infection, gave the highest yield of rhTPO.
