欧洲型PRRSV、PCV2重组痘病毒疫苗灌注式生物反应器培养工艺的建立

Establishment of a technique for perfusion bioreactor culture of a recombinant vaccinia virus against EU PRRSV and PCV2

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。

Objectives To use microcarriers and a bioreactor to culture BHK cells and to inoculate those cells with the recombinant vaccinia virus rVTT-ORF2-EU-RF3O5 in order to establish a process for large-scale preparation of a recombinant vaccinia virus against Eu-PRRSV and PCV2. Methods Perfusion culture was performed using a working volume of 2Lto culture BHK cells.Culture parameters were a temperature of 37℃,a dissolved oxygen（DO）concentration of60%,a mixing speed of 80r/min,and a pH of 7.3.BHK cells were inoculated with the recombinant vaccinia virus rVTT-ORF2-EU-RF3O5 at a dose of 0.1 MOL when 90% of the microcarrier surface covered the cells.During viral infection,the parameters were a culturing temperature of 35℃,a DO concentration of 50%,a mixing speed of 50r/min,and a pH of 7.1. Results Culturing for 96 hyielded a total of 1.587 × 1010 cells,so the cell count increased 39.67-fold.During viral multiplication,the viral titer was 2.12×109 PFU/ml when cells completely separated from the microcarriers. Conclusion A technique for production of a recombinant vaccinia virus against EU PRRSV and PCV2 via perfusion culture was successfully established,providing the technical parameters for the large-scale preparation of a recombinant vaccinia virus vaccine.