| 悬浮MDCK细胞的驯化与H5亚型禽流感病毒的培养 |
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| 引用本文： | 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.悬浮MDCK细胞的驯化与H5亚型禽流感病毒的培养[J].中国农业科学,2018,51(17):3405-3414. |
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| 作者姓名： | 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 |
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| 作者单位： | 吉林冠界生物技术有限公司;中国农业科学院;新疆维吾尔自治区畜牧科学院 |
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| 基金项目： | 陈宏，E-mail：tcswll@163.com。 |
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| 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细胞增殖的病毒毒价相当。细胞源三价灭活疫苗免疫海兰褐商品蛋鸡,首免后21 d时,禽流感H5亚型Re-6株、Re-7株、Re-8株HI抗体效价几何平均值(GMT)分别为1﹕446、1﹕111、1﹕416,二免后30 d时三者HI抗体效价几何平均值(GMT)分别为1﹕588、1﹕362、1﹕776,6个月时分别为1﹕239、1﹕128、1﹕223,维持了较高的抗体水平;免疫长白飞鸭,首免后14 d时,禽流感H5亚型Re-6株、Re-7株、Re-8株HI抗体效价几何平均值(GMT)分别为1﹕30、1﹕17、1﹕64,28 d时三者HI抗体效价几何平均值(GMT)分别为1﹕194、1﹕91、1﹕137,42 d时分别为1﹕416、1﹕128、1﹕239;以上两组的试验结果与鸡胚源重组禽流感灭活疫苗免疫后诱导产生的HI抗体效价相当。【结论】经驯化获得的可在无血清培养基中悬浮生长的MDCK细胞株,其增殖重组禽流感病毒H5N1 Re-6株、H5N1 Re-7株、H5N1 Re-8株能力强,且病毒被制备成灭活疫苗免疫海兰褐商品蛋鸡和长白飞鸭可产生较高水平的抗体。为大规模工业化生产禽流感疫苗提供技术支持。
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| 关 键 词： | MDCK细胞 重组禽流感病毒 悬浮培养 免疫效果 /> |
| 收稿时间： | 2018-04-18 |
Domestication of Suspended MDCK Cells and Cultivation of H5 Subtype Avian Influenza Virus |
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| CHEN Hong,YANG Liu,SONG HaiYan,SHI Ying,MENG LingWei,FU ChunJie,Zhang Dan,ZHAO HaiYuan,LI JinXiang,JIANG XiaoMei,ZHANG TianShu.Domestication of Suspended MDCK Cells and Cultivation of H5 Subtype Avian Influenza Virus[J].Scientia Agricultura Sinica,2018,51(17):3405-3414. |
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| Authors: | CHEN Hong YANG Liu SONG HaiYan SHI Ying MENG LingWei FU ChunJie Zhang Dan ZHAO HaiYuan LI JinXiang JIANG XiaoMei ZHANG TianShu |
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| Institution: | 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 830000 |
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| Abstract: | 【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 H5N1 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 brownlayinghens 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 5L bioreactors showed that the cell line was suitable for scale-up production. The cell density was cultured from 1.5×106 cells/ml to 1.0×107 cells/ml in 48-hours. Under the good condition, the cells had a high activity rate, and were individually suspended in the medium. The H5N1 reassortant avian influenza virus were produced in the suspension MDCK cells, Re-6 strain: HA titer 1﹕512, TCID50 107.67/mL, EID50 107.83/0.1mL; Re-7 strain: HA titer 1﹕512, TCID50 107.33/ml, EID50 107.17/0.1mL; Re-8 strain: HA titer 1﹕1024, TCID50 108.5/mL, EID50 108.38/0.1mL. The results was similar to that of the adherent MDCK cells. Cell-derived trivalent inactivated vaccine was used to immunize y-line brown laying hens after 21 days of the first immunization, and the average HI antibody titer of the avian influenza virus H5 subtype strain Re-6, Re-7 and Re-8 in serum was 1﹕446, 1﹕111, and 1﹕416. The average HI antibody titer of the three groups was 1﹕588, 1﹕362, and 1﹕776 at 30 days after secondary immunization, and they were 1﹕239, 1﹕128, and 1﹕223 at 6 months, maintaining high antibody levels. Changbai flying duck were immured, after 14 days of the first immunization, the average HI antibody titer of the avian influenza virus H5 subtype strain Re-6, Re-7 and Re-8 in serum was 1﹕30, 1﹕17, and 1﹕64. The average HI antibody titer of the three groups was 1﹕194, 1﹕91, and 1﹕137 at 28 days, and they were 1﹕416, 1﹕128, and 1﹕239 at 42 days. The experimental results of the above two groups were equivalent to those derived from chicken embryo.【Conclusion】A domesticated MDCK cell line that could be grown in serum-free medium, and were used as host cells to produce reassortant avian influenza virus. Its ability to proliferate reassortant avian influenza virus H5N1strain Re-6, Re-7 and Re-8 was strong. Viruses that were prepared as inactivated vaccines to immunize y-line brownlayinghens and Changbai flying duck produced higher levels of antibodies. This study provided technical support for large-scale industrial production of the avian influenza vaccine. |
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| Keywords: | MDCK cells reassortant avian influenza virus suspension culture immune effect |
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