基于PK15细胞的猪圆环病毒2型全悬浮培养工艺

doi:10.3864/j.issn.0578-1752.2021.06.017

摘要/Abstract

摘要：

【目的】筛选1株适合PCV2病毒生产的悬浮细胞株,摸索PCV2悬浮生产工艺（病毒种毒来源、MOI及收获时间）,为大规模悬浮培养技术制备疫苗提供试验依据。【方法】使用有限稀释法对PK15原细胞稀释后接种于96孔板,每2 d观察细胞生长和形态,待细胞90%长满后,将细胞从96孔板陆续扩至24孔板、12孔板、6孔板,最后到方瓶,筛选3株可贴壁生长的、形态较好的PK15克隆。3株克隆（PK15-1C8、2F11、1E5）按照1×10⁶细胞/mL的密度,直接接种在PK15的无血清培养基中,并置于37℃,5%二氧化碳,120 r/min的摇床培养箱中继续培养,每天监测细胞密度和活率,每3 d传代,使细胞逐渐适应悬浮环境,驯化为可全悬浮、无血清培养的悬浮细胞株;细胞传代稳定并建库后,接种PCV2病毒,通过对比3株悬浮克隆细胞培养PCV2病毒含量的差异,筛选1株克隆细胞,用于生产PCV2;针对不同种毒（来源于贴壁细胞或悬浮细胞）,摸索感染MOI（0.1、0.2、0.5）及收获时间（48、72、96、120h）,确定PCV2最佳生产工艺。【结果】（1）贴壁细胞置于无血清培养基中,适应至第2代时细胞即可呈悬浮生长,连续传至11代,细胞生长稳定,按照1×10⁶/mL接种细胞,细胞生长72h时细胞密度可达到10×10⁶/mL,活率在95%以上,倍增时间为20h左右;（2）3株悬浮细胞使用相同条件,分别接种PCV2病毒后,PK15-1C8克隆细胞的病毒含量可达到10^6.4TCID₅₀/mL,克隆PK15-2F11（10^5.5TCID₅₀/mL）、PK15-1E5（10^5.6TCID₅₀/mL）,3株克隆细胞病毒含量均高于原克隆（10^4.7TCID₅₀/mL）,但PK15-1C8克隆细胞的病毒含量更高且更稳定,确定为后续研究用细胞;（3）使用贴壁细胞来源的种毒（10^6.4TCID₅₀/mL）感染PK15-1C8克隆细胞后,最优工艺为接毒时细胞密度1×10⁶/mL,以0.1MOI接毒,接毒后72h收获,最高病毒含量为10^6.5TCID₅₀/mL。以来源于悬浮细胞的种毒（10^6.3TCID₅₀/mL）感染细胞后,病毒含量较贴壁细胞来源种毒更高,最高可达10^7.3TCID₅₀/mL,其最优工艺为接毒时细胞密度1×10⁶/mL,以0.2MOI接毒,接毒后72h收获。【结论】通过对PK15细胞进行单克隆筛选,驯化悬浮、3株悬浮细胞接种PCV2后病毒含量的对比,确定一株病毒含量最高的悬浮细胞,并以此悬浮细胞为基础进行PCV2生产工艺摸索,建立了全悬浮无血清培养的PK15-1C8细胞增殖PCV2工艺,该工艺首次使用悬浮细胞扩增PCV2病毒为种毒进行接毒,最高病毒含量可达10^7.3TCID₅₀/mL,可用于工厂化疫苗生产。

关键词: PK15细胞, 猪圆环病毒2型, 克隆筛选, 悬浮培养

Abstract:

【Objective】Selected one suspension PK15 cell line which is suitable for PCV2 virus , and then developed the production process for PCV2 vaccines(source of virus, MOI and harvest time ), to provide basic theory and guarantee for large-scale production use suspension culture instead of adherent culture. 【Method】The PK15 primary cells were diluted using the limiting dilution method and seeded in 96-well plates. The cell growth and morphology were observed every 2 days. After 90% of the cells were overgrown, the cells were gradually expanded from 96-well plates to 24-well plates,to 12-well plates, to 6-well plate, and finally to the square flask, three PK15 clones with good morphology that can grow adherently were selected. Three PK15 clones with good morphology that can grow adherently were selected. Three clones (PK15-1C8, 2F11, 1E5) were directly seeding in PK15 serum-free medium at a density of 1×10⁶cells/mL and placed in a shaker incubator at 37 ℃, 5% carbon dioxide, and 120 r/min to continue culture. Monitor the cell density and viability every day, passage every 3 days, make the cells gradually adapt to the suspension environment, the PK15 clone can culture as a fully suspension cell line and can growth well in serum free media. After suspension cells stability passage and cell bank, compared the PCV2 virus content with three suspension clone cells, one clone cell was selected for PCV2 production. For different kinds of viruses (source from adherent cells or suspension cells), explore the infection MOI (0.1, 0.2, 0.5) and harvest time (48, 72, 96, 120h), determine the best production process of PCV2. 【Results】(1)The results showed that when the adherent cells passage to second generation in serum media CD PK15 259, the cells can suspended growth, continuous passage for eleven generation, suspension cells can growth stable, when seeding with 1×10⁶ /mL cells, cells can reach 10×10⁶ /mL cells when growth at 72h, viability rate is above 95%, and the doubling time is about 20h; (2) The three suspension cells use the same condition to infection PCV2 virus, the virus content of PK15-1C8 cloned cells can reach 10^6.4TCID₅₀/mL, clone PK15-2F11 (10^5.5TCID₅₀/mL), PK15-1E5 (10^5.6TCID₅₀/mL), the virus content of the three cloned cells was higher than that of the original clone (10^4.7TCID₅₀/mL), however, the virus content of PK15-1C8 cloned cells is higher and more stable, so it is determined as a cell for follow-up research; (3) After infecting PK15-1C8 cloned cells with seed virus (10^6.4TCID₅₀/mL) from adherent cells, the optimal process is 1×10⁶/mL cell density with 0.1MOI and harvest at 72h the virus content can reach 10^6.5TCID₅₀/mL. After infecting PK15-1C8 cloned cells with seed virus (10^6.3TCID₅₀/mL) from suspension cells, the optimal process is 1×10⁶/mL cell density with 0.2MOI and harvest at 72h the virus content can reach 10^7.3TCID₅₀/mL. 【Conclusion】Through the monoclonal screening of PK15 cells, adapted to suspension cells compared the PCV2 virus content of 3 suspension cells, a suspension cell with the highest virus content was determined, and based on this suspension cell, explored the process of PCV2 virus, and then established the PCV2 fully suspension, serum-free culture process. The SV15-1C8 cell proliferation PCV2 process of full suspension serum-free culture was established. This process used suspension cells to amplify PCV2 virus for seed poisoning for the first time. The highest virus content can reach 107.3TCID50/mL, which can be used for factory vaccine production. According selection sensitive clones and optimized the process for PCV2, can increase the virus titer, and realizes full suspension culture without serum, improved the production process of PCV2, improve the production efficiency, reduce the cost and improve the quality of the production. This process first use suspension cells to amplify the PCV2 virus. The highest virus content can reach 10^7.3TCID₅₀/mL, which can be used for large-scale PCV2 virus production.

Key words: PK15 cells, PCV2, clone selection, suspension culture

王嘉琪,董育红,姜菊玲,钱建宁,魏文涛,宋国亮,焦金波,关新新,姬郭彪,张业炘. 基于PK15细胞的猪圆环病毒2型全悬浮培养工艺[J]. 中国农业科学, 2021, 54(6): 1280-1287.

JiaQi WANG,YuHong DONG,JuLing JIANG,JianNing QIAN,WenTao WEI,GuoLiang SONG,JinBo JIAO,XinXin GUAN,GuoBiao JI,YeXin ZHANG. Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process[J]. Scientia Agricultura Sinica, 2021, 54(6): 1280-1287.

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试验 Experiment		感染培养基 Infection medium	感染时细胞密度 Infection cell density（cells/mL）	感染量 MOI	收获时间 Harvest time （h）	重复次数 Repeat times
贴壁种毒 Virus from adherent cell	感染MOI摸索 Infection MOI explore	接毒培养基A Infection medium A	1.0×10⁶	0.1、0.2、0.5	72	3
贴壁种毒 Virus from adherent cell	收获时间摸索 Harvest time explore		1.0×10⁶	0.1	48、72、96、120	3
悬浮种毒 Virus from suspension cell	感染MOI摸索 Infection MOI explore	接毒培养基A Infection medium A	1.0×10⁶	0.1、0.2、0.5	72	5
悬浮种毒 Virus from suspension cell	收获时间摸索 Harvest time explore		1.0×10⁶	0.2	48、72、96	5