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首页> 外国专利> A method of obtaining a concentrate of recombinant pseudo-adenovirus particles expressing the hemagglutinin gene of influenza virus A / California / 07/2009 (H1N1) or A / California / 07/2009 (H1N1) -like

A method of obtaining a concentrate of recombinant pseudo-adenovirus particles expressing the hemagglutinin gene of influenza virus A / California / 07/2009 (H1N1) or A / California / 07/2009 (H1N1) -like

机译：一种获得表达流感病毒A / California / 07/2009（H1N1）或A / California / 07/2009（H1N1）样血细胞凝集素基因的重组伪腺病毒颗粒的方法

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A method of obtaining a concentrate of recombinant pseudo-adenovirus particles expressing the A / California / 07/2009 (H1N1) or A / California / 07/2009 (H1N1) -like influenza hemagglutinin gene, similar, characterized in that the technological steps include the following sequences: a bank for a production cell culture — a working bank for a production suspension cell culture with a concentration of 2.0 × 10 cells / ml with an increase in cell yield of not less than 700 times, and obtaining with an increase in active RPAN units by at least 33 times from an RPAN master bank - RPAN working bank with a titer of at least 2 × 10 PFU / ml; - preparation of samples of a disaggregated starting RPAN culture with activity of at least 2 × 10 PFU / ml from the RPAN working bank obtained at the previous stage by purification from hemagglutinins expressed by RPAN during the cultivation of the working RPAN can; - conducting a one-round infection of a production suspension cell culture with a cell concentration of 1.0 × 10cl / ml in a bioreactor with a nutrient medium by introducing a disaggregated starting production culture of RPAN with t thromium of at least 2 × 10 PFU / ml, resulting in an RPAN-containing suspension of crude with a titer of at least 10 PFU / ml by culturing for 24 hours with an increase in the yield of active RPAN units in the process of growing by at least 10 times, with further cell precipitation mass by centrifugation and removal of the spent nutrient medium; - resuspension of the cell mass precipitated at the previous stage in a lysis buffer containing Triton X-100 nonionic detergent at a concentration of 1% for 3 hours, then freezing once Contents for 2 hours, followed cryostorage
机译：一种获得表达类似A / California / 07/2009（H1N1）或A / California / 07/2009（H1N1）样流感血凝素基因的重组伪腺病毒颗粒的方法，其特征在于，所述技术步骤包括按以下顺序排列：生产细胞培养库-生产悬浮细胞培养库，其浓度为2.0×10个细胞/ ml，细胞产量增加不少于700倍，并且获得从RPAN主库-RPAN工作库中滴定至少2×10 PFU / ml的活性RPAN单位至少33次; -从上一阶段获得的RPAN工作库中制备活性至少为2×10 PFU / ml的分解后的RPAN培养样品，方法是在工作RPAN罐头培养过程中从RPAN表达的血凝素中纯化而来; -通过引入具有至少2×10 PFU / t t的RPAN的分解起始生产培养物，在具有营养培养基的生物反应器中对细胞浓度为1.0×10cl / ml的生产悬浮细胞培养物进行一轮感染。通过进一步培养至少24个小时，通过培养24小时使活性RPAN单元的产量增加至少10倍，从而得到滴度至少为10 PFU / ml的含RPAN的原油混悬液通过离心和去除用过的营养培养基产生沉淀物; -将上一阶段沉淀的细胞团重悬浮在含有Triton X-100非离子去污剂的裂解缓冲液中，其浓度为1％3小时，然后冷冻一次，将内容物冷冻2小时，然后冷冻

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