摘要
【目的】为评估细胞源重组禽流感病毒H5亚型灭活疫苗的有效性提供数据支持。【方法】选取一株来源清楚的贴壁MDCK细胞,通过逐步降低培养基中血清含量及不断调整无血清培养基配方,将其驯化为悬浮MDCK细胞,并以此为基质增殖重组禽流感病毒H5N1 Re-6株、H5N1 Re-7株和H5N1 Re-8株;比较不同毒株在贴壁和悬浮MDCK细胞中增殖的差异。分别将经悬浮MDCK细胞增殖的病毒与经SPF鸡胚增殖的病毒制备成重组禽流感病毒(H5亚型)三价灭活疫苗,免疫商品蛋鸡和商品鸭,通过血清学方法比较细胞源与鸡胚源禽流感灭活疫苗的免疫效果。海兰褐商品蛋鸡:6 050只,分为3组,2组为免疫组,每组3 000只,28日龄免疫0.5 mL/只,80日龄免疫0.5 mL/只;不免疫对照组1组,50只,同等条件下隔离饲养。分别于蛋鸡日龄49、110、210 d(即首免后21 d、82 d、6个月)时采血分离血清,测定禽流感H5亚型Re-6株、Re-7株、Re-8株的HI抗体效价,监测抗体消长。长白飞鸭:220只,分为3组,2组为免疫组,每组100只,10日龄免疫0.5 mL/只,24日龄免疫1.0 mL/只,不免疫对照组20只,同等条件下隔离饲养。分别于鸭日龄24、38、52 d(即首免后14、28、42 d)时采血分离血清,测定禽流感H5亚型Re-6株、Re-7株、Re-8株的HI抗体效价,监测抗体消长。【结果】获得一株可在无血清培养基中悬浮生长的MDCK细胞,摇瓶、5L生物反应器中的培养数据及细胞状态显示,该细胞株适合放大生产。此悬浮MDCK细胞培养48 h细胞密度可由1.5×10~6 cells/mL左右增殖到1.0×10~7 cells/mL左右,状态良好、活率高、单个悬浮于培养基中。以此悬浮MDCK细胞为基质增殖重组禽流感病毒,H5N1 Re-6株的HA效价达1﹕512,TCID_(50) 10^(7.67)/mL,EID_(50) 10^(7.83)/0.1 mL;H5N1 Re-7株的HA效价达1﹕256,TCID_(50)达10^(7.33)/mL,EID_(50)10^(7.17)/0.1 mL;H5N1 Re-8株的HA效价达1﹕1024,TCID_(50) 10^(8.5)/mL,EID_(50) 10^(8.38)/0.1 mL,与经贴壁MDCK细�
【Objective】This study provided data support for assessing the effectiveness of cell-derived reassortant influenza virus H5 subtype inactivated vaccines.【Method】 A clearly defined MDCK cells was selected to be domesticated to suspend MDCK cells, and the reassortant avian influenza virus H5 N1 Re-6 strain, Re-7 strain and Re-8 strain were proliferated in it. The proliferation differences of different strains in the adherent and suspended MDCK cells were compared. The reassortant avian influenza virus（H5 subtype） trivalent inactivated vaccine were prepared by suspended MDCK cells and by SPF chicken embryo, respectively, which were immuned in commercial chicken and commercial duck. Immune effects were compared between cell source and chicken embryo source by serological methods. 6 050 Hy-line brown laying hens were divided into 3 groups： 2 groups were immunized groups, 3 000 hens in each group, immunized 0.5 mL/head at 28 days of age and 80 days of age; 1 non-immunized control group, 50 hens were reared under the same conditions. The serums were collected from the layers at 49 days, 110 days, and 210 days（ie, 21 days, 82 days, and 6 months after the first immunization） to determine the reassortant avian influenza virus H5 subtype Re-6 strain, Re-7 strain and Re-8 strain, The HI antibody titer was monitored. 220 Changbai flying ducks were divided into 3 groups： 2 groups were immunized groups, 100 ducks in each group, immunized 0.5 mL/head at 10 days of age, immunized 1.0 mL/head at 24 days of age; 20 ducks in non-immunized control group, under the same conditions. The serums were collected at 24, 38, and 52 days（ie, 14 days, 28 days, and 42 days after the first immunization） to determine the reassortant avian influenza virus H5 subtype Re-6 strain, Re-7 strain and Re-8 strain, and the HI antibody titer was monitored. 【Result】A MDCK cell that could be grown in serum-free medium was obtained. Culture data, cell status in shake flasks and 5 L bioreactors showed that the cell line was suitable
作者
陈宏
杨柳
宋海岩
石莹
孟令伟
付春杰
张丹
赵海源
李金祥
蒋晓梅
张天舒
CHEN Hong1, YANG Liu1, SONG HaiYan1, SHI Ying1, MENG LingWei1, FU ChunJie1, ZHANG Dan1, ZHAO HaiYuanl, LI JinXiang2, JIANG XiaoMei3, ZHANG TianShu3(1.Jilin Guanjie Biotechnology Co. LTD, Meihekou 135000, Jilin; 2.Chinese Academy of Agricultural Sciences, Beijing 100081; 3.The Xinjiang Uygur Autonomous Region Academy of Animal Science, Urumqi 83000)
出处
《中国农业科学》
CAS
CSCD
北大核心
2018年第17期3405-3414,共10页
Scientia Agricultura Sinica
关键词
MDCK细胞
重组禽流感病毒
悬浮培养
免疫效果
MDCK cells
reassortant avian influenza virus
suspension cutture
immune effect
