Vero细胞培养A/Shanghai/CN02/2013(H7N9)Va流感病毒适宜条件研究
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摘要
目的:优化Vero细胞培养H7N9流感病毒的培养条件,建立细胞培养病毒高产培养系统。方法:采用不同病毒接种MOI、培养温度、pH值和TPCK胰酶等条件优化培养H7N9流感病毒Vero细胞适应株A/Shanghai/CN02/2013(H7N9)Va的条件,并将其连续传代后扩大至细胞工厂大规模培养。结果:Vero细胞高产H7N9流感病毒培养条件为DMEM/F12和MEM以1∶1混合,并添加0.3 mg/mL牛血清白蛋白、1%谷氨酰胺、0.5%双抗和1.5μg/mL TPCK胰蛋白酶,pH为7.4时,连续传代病毒血凝效价保持在512,扩大至细胞工厂大规模培养病毒产量稳定。结论:建立的Vero细胞培养H7N9流感病毒条件,能够持续稳定高产,并适用于细胞工厂大规模培养,有望用于H7N9流感细胞疫苗的大规模制备。
Objective: To optimize the culture conditions for Vero cell cultured H7 N9 influenza virus and establish a high-yield culture system. Methods: Under different virus inoculation MOI, culture temperature, pH and concentration of TPCK-trypsin, the H7 N9 Vero cell-adapted strain A/Shanghai/CN02/2013(H7 N9)Va were cultured and passaged to the cell factory scale. Results: The optimal virus culture conditions for H7 N9 influenza virus high-yield Vero cell were DMEM/F12 and MEM mixed at a ratio of 1∶1, and addition of 0.3 mg/mL BSA, 1%glutamine, 0.5% penicillin and streptomycin and 1.5 μg/mL TPCK-trypsin at pH7.4, under which, the continuous passage of virus hemagglutination titer reached and remained 512, till large-scale of cell factories and stable virus production. Conclusion: The optimal conditions for H7 N9 influenza virus cultured in Vero cells is expected to be used in large-scale production of H7 N9 cell influenza vaccine.
Objective: To optimize the culture conditions for Vero cell cultured H7 N9 influenza virus and establish a high-yield culture system. Methods: Under different virus inoculation MOI, culture temperature, pH and concentration of TPCK-trypsin, the H7 N9 Vero cell-adapted strain A/Shanghai/CN02/2013(H7 N9)Va were cultured and passaged to the cell factory scale. Results: The optimal virus culture conditions for H7 N9 influenza virus high-yield Vero cell were DMEM/F12 and MEM mixed at a ratio of 1∶1, and addition of 0.3 mg/mL BSA, 1%glutamine, 0.5% penicillin and streptomycin and 1.5 μg/mL TPCK-trypsin at pH7.4, under which, the continuous passage of virus hemagglutination titer reached and remained 512, till large-scale of cell factories and stable virus production. Conclusion: The optimal conditions for H7 N9 influenza virus cultured in Vero cells is expected to be used in large-scale production of H7 N9 cell influenza vaccine.
引文
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[2]Herfst S,Imai M,Kawaoka Y,et al.Avian influenza virus transmission to mammals[M]//Influenza pathogenesis and control.Volume I.Springer International Publishing,2014:137-155.
[3]Kile J C,Ren R,Liu L,et al.Update:Increase in human infections with novel Asian lineage avian influenza A(H7N9)viruses during the fifth epidemic-China,October 1,2016-August 7,2017[J].MMWR Morb Mortal Wkly Rep,2017,66(35):928.
[4]Johansson B E,Cox M M.Influenza viral neuraminidase:the forgotten antigen[J].Expert Rev Vaccines,2011,10:1683-1695.
[5]Le Ru A,Jacob D,Transfiguracion J,et al.Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing[J].Vaccine,2010,28:3661-3671.
[6]Belsey M,Evans D,Pavlou A,et al.Growth drivers and resistors of the influenza market:the importance of cell culture flu[J].J Commer Biotechnol,2006,12:150-155.
[7]Guerra A,Koldovsky U,Henle W.Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs[J].Virology,2003,313(2):473-480.
[8]Barrett P N,Mundt W,Kistner O,et al.Vero cell platform in vaccine production:moving towards cell culture-based viral vaccines[J].Exp Rev Vaccines,2009,8:607-618.
[9]郭琦.H7N9大流行流感疫苗(Vero细胞)毒株生物学特性及其制备工艺研究[D].北京:北京协和医学院,2018.
[10]Job E R,Bottazzi B,Gilbertson B,et al.Serum amyloid P is a sialylated glycoprotein inhibitor of influenza A viruses[J].PLoS One,2013,8(3):e59623.
[11]马磊,耿兴良,苏杨起,等.用无血清适应的Vero细胞培养H5N1型流感病毒条件的优化[J].中国生物制品学杂志,2015,28(7):741-745.
[12]谢英林,周庆玲,王文军,等.流感病毒H5N1在Vero细胞上培养条件的优化[J].中国生物制品学杂志,2015,28(10):1089-1091.