全悬浮驯化MDBK细胞应用于牛病毒性腹泻、牛传染性鼻气管炎二联灭活疫苗的效果评估  

Efficacy Evaluation of BVDV and IBRV Inactivated Vaccine using Completely Suspended MDBK Cells

作　　者：杨青春 徐凌[2] 刘国英 范秀丽 王秉昆 王凯 李晓燕 李超 田志辉 陈坚 赵丽霞 宋庆庆 YANG Qing-chun;XU Ling;LIU Guo-ying;FAN Xiu-li;WANG Bing-kun;WANG Kai;LI Xiao-yan;LI Chao;TIAN Zhi-hui;CHEN Jian;ZHAO Li-xia;SONG Qing-qing(Spirit Jinyu Biological Pharmaceutical Co.,Ltd.,Hohhot 010030,China;Affiliated Hospital of Inner Mongolian Medical University,Hohhot 010050,China)

机构地区：[1]金宇保灵生物药品有限公司,内蒙古呼和浩特010030 [2]内蒙古医科大学附属医院,内蒙古呼和浩特010050

出　　处：《中国兽医杂志》2022年第10期74-81,共8页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划资助(2017YFD0500903)。

摘　　要：为了给规模化生产牛病毒性腹泻病毒(BVDV)和牛传染性鼻气管炎病毒(IBRV)提供悬浮细胞培养方法,本试验采用4种降血清方法,使贴壁MDBK细胞逐步适应了悬浮培养环境,最终获得了1株MDBK悬浮细胞株,同时摸索出MDBK悬浮细胞株培养BVDV和IBRV抗原的最佳参数;将MDBK悬浮细胞株扩大到60 L和500 L后连同MDBK贴壁细胞一同接种BVDV和IBRV,比较3种细胞培养后的病毒滴度;通过使用悬浮培养方法制备BVDV-IBRV二联灭活疫苗,按照2.00、1.00、0.50 mL/头和0.25 mL/头4个剂量免疫健康牛,确定其攻毒保护效果,继而免疫不同年龄黑白花奶牛群体(包括犊牛、后备母牛和成年母牛),检测免疫后牛群的BVDV和IBRV中和抗体效价。结果显示:在60 L和500 L规模化放大培养过程中,IBRV病毒滴度各为8.25 lgTCID_(50)/mL和8.00 lgTCID_(50)/mL,BVDV病毒滴度各为8.00 lgTCID_(50)/mL和7.75 lgTCID_(50)/mL,与MDBK贴壁细胞株产毒毒价几乎相同;免疫攻毒保护试验中,最低免疫剂量为1.0 mL/头,免疫保护率能在80%以上;二联灭活苗免疫的牛群试验中,IBRV和BVDV中和抗体几何平均值均显著高于未免疫组,达到了免疫保护效果。结果表明,本试验成功筛选到1株MDBK悬浮培养细胞株,并且通过悬浮培养工艺制备的BVDV-IBRV二联灭活疫苗免疫效果良好,该MDBK细胞全悬浮培养株的成功驯化为疫苗规模化制备提供了科学依据。This study aimed to obtain completely suspended cells for large-scale antigen production from bovine viral diarrhea virus(BVDV)and infectious bovine rhinotracheitis virus(IBRV).At four decreasing serum concentrations,adherent Madin-Darby bovine kidney(MDBK)cells adapted to suspension culture gradually,and became completed suspended.At the same time,the optimal parameters for culturing BVDV and IBRV antigens in MDBK suspension cell lines were explored.MDBK suspension cells were then expanded to 60 L and 500 L and inoculated with BVDV and IBRV,the virus titers of the three cell cultures were compared.The inactivated BVDV-IBRV vaccine prepared from suspension-cultured antigens was administered to healthy cattle with 2.00,1.00,0.50 mL/head and 0.25 mL/head to determine its protective effect,and subsequently given to different aged holstein cows,including calves,virgin cows and adult cows to detect the neutralizing antibody titers of BVDV and IBRV.The results showed that IBRV had antigen titers of 8.25 lgTCID 50/mL and 8.00 lgTCID 50/mL,respectively,and BVDV had antigen titers of 8.00 lgTCID 50/mL and 7.75 lgTCID 50/mL,respectively,from 60 L and 500 L productions,the same as those produced by adherent MDBK cells.Immune challenge protection studies showed that the minimum immune dose was 1.0 mL/head,and the immune protection rate was above 80%.The geometric mean of IBRV and BVDV neutralizing antibody of cattle with dual inactivated vaccine immunizations was significantly higher than that of the non-immunized group.Thus,MDBK suspension cells supported large-scale antigen production,and the inactivated BVDV-IBRV vaccine prepared from MDBK suspension culture had strong immune effect.The successful domestication of the MDBK cell suspension culture strain provides a scientific basis for the large-scale preparation of vaccines.

关 键 词：牛病毒性腹泻病毒 牛传染性鼻气管炎病毒 MDBK细胞 悬浮培养 免疫效果 

分 类 号：S859.79[农业科学—临床兽医学]