基于生物反应器的猪流行性腹泻病毒(ZJ/15株)悬浮培养工艺研究

Study on suspension culture technology of Porcine epidemic diarrhea virus(ZJ/15 strains)based on bioreactor

为了优化生物反应器全悬浮培养技术制备猪流行性腹泻病毒(ZJ/15株)的工艺,试验首先通过摇瓶培养对病毒接种时的细胞密度、胰酶浓度、接毒剂量和收毒时间等培养条件进行优化;之后按照摇瓶培养确认的工艺进行生物反应器全悬浮培养工艺验证,同时对pH值、溶氧值(DO)、转速等培养参数进行优化,以病毒含量(TCID_(50))为指标,最终筛选出在15L生物反应器中培养猪流行性腹泻病毒(ZJ/15株)的最优条件,并进一步在50L生物反应器中进行工艺验证,将病毒液灭活后稀释至1×10^(7).0TCID_(50)/mL免疫怀孕75~90d的健康易感初怀母猪,在分娩当日,采集母猪和仔猪血清,并对分娩的3日龄仔猪攻毒进行免疫原性试验。结果表明:猪流行性腹泻病毒(ZJ/15株)在摇瓶上的最适培养条件为当细胞密度达到6×10^(6)个/mL以上时,用含胰酶(终浓度为20μg/mL)的无血清培养基将细胞密度稀释至2×10^(6)个/mL,按感染复数(MOI)=0.1接毒,130 r/min摇床振荡培养36h可收获病毒液,病毒含量可稳定达到1×10^(7.5)TCID_(50)/mL以上;猪流行性腹泻病毒(ZJ/15株)在15L生物反应器上的最适培养条件为pH值7.0~7.2、DO值50%、转速80~100 r/min,培养36h收获的病毒液含量可稳定在1×10^(7.5)TCID_(50)/mL以上;在50L生物反应器上放大培养收获的病毒液含量为1×10^(7).8TCID_(50)/mL。母猪和仔猪中和抗体效价均≥1∶32,免疫保护力为100%。说明本试验条件下成功建立并优化了生物反应器全悬浮培养制备猪流行性腹泻病毒(ZJ/15株)的工艺,生产的猪流行性腹泻病毒(ZJ/15株)的免疫原性较好。

In order to optimize the preparation process of porcine epidemic diarrhea virus(ZJ/15 strain)by total suspension culture in biorereactor,in the experiment,the cell density,pancreatic enzyme concentration,dose and time of inoculation were optimized by shaking flask culture.After that,the full suspension culture process of the bioreactor was verified according to the process confirmed by shaking flask culture.Meanwhile,the culture parameters such as pH value,dissolved oxygen value(DO)and rotational speed were optimized,and the virus content(TCID50)was used as the index to finally select the optimal conditions for the culture of porcine epidemic diarrhea virus(ZJ/15 strain)in a 15L bioreactor.The process was further verified in a 50 L biorereactor,and the venom was inactivated and diluted to 1×10^(7.0)TCID_(50)/mL of healthy susceptible first pregnant sows during 75-90 days of immune pregnancy.On the day of delivery,serum of sows and piglets was collected,and immunogenicity test was performed on 3-day-old piglets after delivery.The results showed that when the cell density of porcine epidemic diarrhea virus(ZJ/15 strain)reached more than 6×10^(6)cells/mL,the cell density was diluted to 2×10^(6)cells/mL with serum-free medium containing pancreatic enzyme(final concentration of 20μg/mL),and the cell density was inoculated according to the number of infections(MOI)=0.1.After 36 h of shaking culture in a 130 r/min shaker,the venom could be harvested,and the virus content could reach more than 1×10^(7.5)TCID_(50)/mL.The optimal culture conditions of porcine epidemic diarrhea virus(ZJ/15 strain)in a 15-L bioreactor were pH 7.0-7.2,DO 50%,and rotation speed 80-100 r/min,and the venom content of 36 h was stable above 1×10^(7.5)TCID_(50/)mL.The venom content was 1×10^(7.8)TCID_(50)/mL in 50 L bioreactor.The titer of neutralizing antibody in sows and piglets was≥1:32,and the immune protection was 100%.The results indicated that the preparation process of porcine epidemic diarrhea virus(ZJ/15 strain)was successfully established and optimized under the conditions of this experiment,and the immunogenicity of the produced porcine epidemic diarrhea virus(ZJ/15 strain)was good.