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目的利用微载体-生物反应器技术培养乳仓鼠肾细胞,接种重组痘病毒rVTT-ORF2-EUO3O5,建立规模化制备欧洲型PRRSV、PCV2重组痘病毒疫苗的灌注式生产工艺。方法利用2L体积灌注式生物反应器培养BHK细胞,设定培养参数为温度37℃、溶氧度(DO)为60%,转速为80r/min,pH值为7.3。当90%微载体表面贴满BHK细胞时以0.1MOI的感染剂量接种重组痘病毒rVTT-ORF2-EUO3O5,设定感染参数为温度35℃、DO为50%、转速为50r/min,pH值为7.1,继续培养观察。结果在采用灌注式生物反应器技术制备重组痘病毒过程中的细胞培养阶段,细胞培养96h可获得1.587×1010个细胞数,细胞增殖39.67倍;在病毒增殖阶段,微载体上细胞完全脱落时的病毒滴度为2.12×109 PFU/ml。结论成功建立了欧洲型PRRSV、PCV2重组痘苗病毒灌注式生产工艺,为规模化制备重组痘病毒疫苗提供了技术参数。
OBJECTIVE: To culture rabbit hamster kidney cells with microcarrier-bioreactor technology and inoculate recombinant poxvirus rVTT-ORF2-EUO3O5 to establish a perfusion-type production process for large-scale preparation of European-type PRRSV and PCV2 recombinant poxvirus vaccine. Methods BHK cells were cultured in a 2 L volumetric bioreactor. The culture parameters were set as follows: temperature 37 ℃, DO 60%, rotation speed 80 r / min, pH 7.3. When 90% of microcarriers were surface-coated with BHK cells, the recombinant poxvirus rVTT-ORF2-EUO3O5 was inoculated at an infection dose of 0.1 MOI. The infection parameters were set as follows: temperature 35 ℃, DO 50%, rotation speed 50 r / min, pH value 7.1, continue to develop observation. Results In the stage of cell culture during the preparation of recombinant poxvirus by the perfusion bioreactor technology, the number of cells reached 1.587 × 10 ^ (-1) after culturing for 96h and the cell proliferation reached 39.67 times. During the stage of virus proliferation, The virus titer was 2.12 × 109 PFU / ml. Conclusion The perfusion-type production process of European-type PRRSV and PCV2 recombinant vaccinia virus was successfully established, which provided the technical parameters for the large-scale preparation of recombinant poxvirus vaccine.