BHK-21细胞无血清悬浮培养生产新城疫病毒

为建立基于无血清悬浮培养细胞生产新城疫病毒(NDV)的工艺,本研究首先筛选了适于NDV增殖的乳仓鼠肾细胞(BHK-21)单克隆细胞株,并将鸡胚适应的NDV在筛选获得的细胞株(BHK-v002)中传代,获得细胞适应的NDV。进一步采用单因素实验法检测病毒感染复数(MOI)、TPCK-胰酶浓度、细胞培养液的稀释比例等工艺参数对病毒效价的影响。结果显示,NDV在无血清培养的BHK-v002细胞中增殖的最适条件为:当细胞生长至约9.0×10^6个/mL时,以培养液.新鲜培养基为2:1的比例补加新鲜培养基,使细胞密度达6.0×10^6个/m L,按MOI为0.005接种NDV LaSota株,TPCK-胰酶终浓度为5μg/mL。接种病毒后96 h收获病毒液的HA效价为8.5 log2HAU/25μL,单细胞产毒量(Svy)达到1 685.9病毒颗粒/细胞,半数组织细胞感染剂量(TCID50)为7.9 log10TCID50/100μL。本研究确定了NDV LaSota株在BHK-21细胞悬浮培养中的增殖条件,建立了基于BHK-21细胞无血清悬浮培养体系中NDV的生产工艺,该工艺操作简便,易于放大,为当前ND疫苗的鸡胚生产工艺提供了候选替代方案。

Production of Newcastle disease virus by serum-free suspension culture of BHK-21 cells

In order to develop the process for producing new castle disease virus(NDV)based on the serum-free suspension culture,in this study,a monoclonal cell clone of baby hamster kidney cells(BHK-v002)was screened for the NDV productivity.The chicken embryo-adapted NDV was passaged in the selected BHK-v002 cell line to obtain cell-adapted NDV.Further,the process parameters such as multiplicity of infection(MOI),TPCK-trypsin concentration and dilution ratio of cell culture mediumon virus titer was tested by single factor experiments.The results showed that the optimal NDV propagating conditions in serum-free BHK-v002 cells were:the cells were diluted into 6.0×106 cells/mL by supplemented with fresh mediumat a ratio of 2:1 in culture medium when the cell density reached to about 9.0×106 cells/mL.The diluted cells supplemented with TPCK-trypsin at a final concentration of 5μg/mL were infected with NDV La Sota at 0.005 MOI.Under these conditions,after 96 hours of virus inoculation the HA titer was 8.5 log2HAU/25μL,and the cell-specific virus yield(Svy)and the half-cell tissue infective dose(TCID50)were 1 685.9 virions/cell and 7.9 log10TCID50/100μL,respectively.This study determined the proliferation conditions of NDV LaSota strain in suspension culture of BHK-21 cells, and established a NDV production process based on the serum-free suspension culture system of BHK-21 cells. The process is simple and easy to scale up, and provides a candidate alternative to the current chicken embryo production process for ND vaccines.