BHK-21悬浮细胞培养条件筛选及口蹄疫病毒悬浮培养工艺研究  

Screening of BHK-21 suspension cell culture conditions and study on suspension culture technology of Foot-and-mouth disease virus

作　　者：施程洪 陆礼琼 李彦荣 吴海燕 段春平 李应福 赵路路 兰虎云 彭正啟 王继华 刘术新 SHI Chenghong;LU Liqiong;LI Yanrong;WU Haiyan;DUAN Chunping;LI Yingfu;ZHAO Lulu;LAN Huyun;PENG Zhengqi;WANG Jihua;LIU Shuxin(Zhongpu Biopharmaceutical Co.,Ltd.,Baoshan 678000,China)

机构地区：[1]中普生物制药有限公司,云南保山678000

出　　处：《黑龙江畜牧兽医》2022年第18期119-129,共11页Heilongjiang Animal Science And veterinary Medicine

摘　　要：为了实现在幼地鼠肾细胞(BHK-21细胞)低血清全悬浮培养工艺下获得更高含量的口蹄疫病毒(FMDV),试验对比了4株不同来源的BHK-21悬浮细胞的培养特性,按体积1%、2%和5%分别接种O型、A型FMDV,接种后4,8,12,16,20,24小时取样进行146S、半数致死量(LD)对比研究,筛选形态良好、培养特性更稳定的BHK-21悬浮细胞株;以摇瓶悬浮培养筛选得到的BHK-21悬浮细胞株,使用O型FMDV O/HB/HK/99株、A型FMDV AF/72株,从接种剂量、活细胞密度(VCD)、病毒培养温度、病毒培养pH值、换液体积比例等工艺参数对病毒146S表达的影响进行了研究,并在7.5,50,500 L生物反应器中逐级放大,进行了O型FMDV O/HB/HK/99株、A型FMDV AF/72株各3个批次病毒液悬浮培养研究,确定O型FMDV O/HB/HK/99株、A型FMDV AF/72株接种BHK-21悬浮细胞的培养工艺。结果表明:筛选得到1株形态良好、培养特性更稳定的BHK-21-CP005悬浮细胞株;O型FMDV O/HB/HK/99株、A型FMDV AF/72株在摇瓶低血清悬浮培养的BHK-21-CP005悬浮细胞株中增殖的最适条件为,当细胞培养48 h, VCD达4.5×10~6/mL,按体积2%接种O/HB/HK/99株和AF/72株,培养温度为36.5~37.0℃,pH值为7.30~7.50,按4∶5体积比例换液,接种病毒16~20 h后收获病毒液,病毒146S含量不低于9.0μg/mL,LD不低于1×10/0.2 mL。说明使用BHK-21-CP005株低血清全悬浮培养生产更高含量的FMDV工艺稳定,可以实现规模化生产。In order to obtain a higher content of Foot-and-mouth disease virus(FMDV) under the low-serum whole suspension culture of BHK-21 cells, the culture characteristics of 4 strains of BHK-21 suspension cells from different sources were compared. O-type and A-type FMDV were inoculated at 1%, 2% and 5% of the volume, respectively, and samples were taken at 4, 8, 12, 16, 20, and 24 h after inoculation for comparative study of 146 S and median lethal dose(LD). BHK-21 suspension cell line with good morphology and more stable culture characteristics was screened;BHK-21 suspension cell line was screened by shake flask suspension culture. Using O-type FMDV O/HB/HK/99 strain and A-type FMDV AF/72 strain, the study was carried out according to the effect of inoculation dose, viable cell density(VCD), virus culture temperature, virus culture pH value, medium exchange ratio and other process parameters on virus 146 S expression. And in the 7.5,50,500 L bioreactors, it was amplified step by step, and 3 batches of virus suspension culture of O-type FMDV O/HB/HK/99 strain and A-type FMDV AF/72 strain were carried out;the culture process of O-FMDV O/HB/HK/99 strain and A-FMDV AF/72 strain inoculated with BHK-21 suspension cells was determined. The results showed that a BHK-21-CP005 suspension cell line with good morphology and more stable culture characteristics was screened out. The optimal conditions for the proliferation of O-type FMDV O/HB/HK/99 strain and A-type FMDV AF/72 strain in BHK-21-CP005 suspension cell line cultured in low serum suspension in shake flasks were as follows: when the cells were cultured for 48 h, the VCD reached 4.5 ×10~6/mL;O/HB/HK/99 strain and AF/72 strain were inoculated at 2% by volume;the culture temperature was 36.5-37.0 ℃;the pH value was 7.30-7.50;the medium was changed by 4∶5 in volume;the virus solution was harvested 16-20 hours after virus inoculation;the content of virus 146 S was not lower than 9.0 μg/mL, and the LDwas not lower than 1×10/0.2 mL. The results suggested that the use of

关 键 词：口蹄疫病毒 BHK-21 低血清 反应器 悬浮培养 146S 半数致死量 

分 类 号：S852.65[农业科学—基础兽医学] S859.53[农业科学—兽医学]