| 生物反应器培养BHK-21悬浮细胞增殖伪狂犬病病毒工艺优化 |
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| 引用本文： | 王家敏,李自良,马芳芳,康碧静,田玲,李倬,马忠仁,乔自林.生物反应器培养BHK-21悬浮细胞增殖伪狂犬病病毒工艺优化[J].畜牧兽医学报,2022,53(11):3936-3947. |
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| 作者姓名： | 王家敏 李自良 马芳芳 康碧静 田玲 李倬 马忠仁 乔自林 |
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| 作者单位： | 1. 西北民族大学 生物医学研究中心 甘肃省动物细胞技术创新中心, 兰州 730030;2. 四川大学 生物治疗国家重点实验室, 成都 610041;3. 西北民族大学 生命科学与工程学院, 兰州 730030 |
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| 基金项目： | 西北民族大学中央高校基本科研业务费专项资金项目（31920210053）；教育部动物医学生物工程创新团队（IRT_17R88） |
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| 摘 要： | 旨在筛选伪狂犬病病毒（PRV）敏感的BHK-21细胞并分析其生长和病毒增殖特性，优化反应器中BHK-21悬浮细胞的培养和病毒增殖条件，建立生物反应器培养BHK-21悬浮细胞增殖PRV工艺。本研究利用响应面和单因素优化法，以细胞生长动力学特性、TCID50病毒滴度等参数为指标，优化1.2 L生物反应器中BHK-21悬浮细胞的最佳培养和增殖病毒条件，在5 L生物反应器中进一步批培养验证。结果显示，筛选获得PRV高敏感的BHK-21-02贴壁细胞和BHK-21-XF02悬浮细胞各1株，BHK-21-XF02悬浮细胞在含3%血清的SLM-BHK低血清培养基和SFM-BHK无血清培养基中均能实现良好的生长和病毒增殖。利用响应面法优化得到1.2 L反应器最佳培养条件为接种密度1.20×106cells·mL-1、搅拌转速120 r·min-1、DO值40%，5 L反应器批培养72 h细胞密度可达（7.61±0.18）×106 cells·mL-1、细胞活率为（96.93±1.18）%。利用单因素法优化得到1.2 L反应器最佳病毒增殖条件为MOI 0.001、培养温度37℃、细胞密度2.0×106cells·mL-1、搅拌转速80 r·min-1，5 L反应器批培养接毒后48 h病毒滴度达到最大值（7.13±0.11） lgTCID50·mL-1。本研究可为PRV疫苗相关研究和规模化生产提供参考。
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| 关 键 词： | BHK-21悬浮细胞 生物反应器 伪狂犬病病毒 敏感性 |
| 收稿时间： | 2022-04-19 |
Optimization of BHK-21 Suspension Cells to Propagate Pseudorabies Virus in Bioreactor |
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| WANG Jiamin,LI Ziliang,MA Fangfang,KANG Bijing,TIAN Ling,LI Zhuo,MA Zhongren,QIAO Zilin.Optimization of BHK-21 Suspension Cells to Propagate Pseudorabies Virus in Bioreactor[J].Acta Veterinaria et Zootechnica Sinica,2022,53(11):3936-3947. |
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| Authors: | WANG Jiamin LI Ziliang MA Fangfang KANG Bijing TIAN Ling LI Zhuo MA Zhongren QIAO Zilin |
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| Institution: | 1. Gansu Tech Innovation Center of Animal Cell, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China;2. The State Key Laboratory of Biotherapy and Cancer Center, Sichuan University, Chengdu 610041, China;3. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China |
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| Abstract: | In this study, we screened the BHK-21 cells which were sensitive to pseudorabies (PRV) and analyzed their growth and virus production characteristics. The conditions of cell culture and virus production in the reactor were optimized, so we finally established the PRV propagation process of BHK-21 suspension cells in bioreactor. The optimal culture and virus propagation conditions of BHK-21 suspension cells in 1.2 L bioreactor based on cell growth dynamics and TCID50 virus titer, were optimized by response surface methodology and single factor optimization method, and further batch culture was carried out in 5 L bioreactor. The results showed that one of adherent cells named BHK-21-02 and one of suspension cells named BHK-21-XF02 with high PRV sensitivity were screened. BHK-21-XF02 suspension cells could achieve good growth and virus propagation in serum-low medium containing 3% serum and serum-free medium. Using response surface methodology, the optimum cell culture conditions of 1.2 L reactor were as follows:inoculation density was 1.20×106 cells·mL-1, stirring speed was 120 r·min-1 and DO was 40%, the cell density could reach (7.61±0.18)×106 cells·mL-1 and the cell viability was (96.93±1.18)% after batch culture in 5 L reactor for 72 hours. Using single factor method, the optimal conditions of virus propagation in 1.2 L reactor were as follows:MOI was 0.001, temperature was 37℃, cell density for virus inoculation was 2.0×106cells·mL-1 and stirring speed was 80 r·min-1. Under conditions, the virus titer reached (7.13±0.11) lgTCID50·mL-1 at 48 hours in 5 L reactor batch culture. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of PRV vaccines. |
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| Keywords: | BHK-21 suspension cell bioreactor pseudorabies virus susceptibility |
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