鸡新城疫病毒LaSota株在BHK-21细胞上增殖条件的优化

doi:10.16431/j.cnki.1671-7236.2019.03.026

Abstract

Abstract:

The aim of this study was to optimize the proliferation conditions of Newcastle disease virus (NDV) LaSota strain on BHK-21 cells,and provide reference for the application of cell line to produce NDV.Firstly,the tolerant concentration of BHK-21 cells to TPCK-trypsin was determined.On this basis,different concentrations of TPCK-pancreatin and newborn bovine serum were added into the medium respectively,and the pH of medium was regulated.The NDV LaSota strain was cultured in the medium and the optimal trypsin concentration,serum concentration and pH of the medium were determined respectively through measuring half-infection amount (TCID₅₀) of the culture.On the other hand,the same quantity of NDV LaSota strain were inoculated to different number of cells or the same number of cells were inoculated by different quantity of virus.The optimal number of BHK-21 cell and the optimal dose of NDV LaSota strain were determined by measuring TCID₅₀.Secondly,the proliferation curve was drawn to determine the optimal time for collection after inoculating the NDV LaSota strain in BHK-21 cells and the TCID₅₀ of the supernatant was determinated at different time.Finally,the NDV LaSota strain was cultured in spinner flask and the TCID₅₀,the half egg infection amount (EID₅₀) and the serum agglutination price (HA) were measured.The results showed that the optimal TPCK-trypsin concentration was 3 μg/mL,the optimal toxic dose was 10^6.375 EID₅₀,the number of cells was 1×10⁶,the pH of the medium was 7.2,and cultured in serum-free medium when the NDV LaSota strain was inoculated in BHK-21 cells.The optimum harvested time was from 34 to 36 h after BHK-21 cells were inoculated with NDV LaSota strain.At the end,the proliferation of NDV LaSota strain was carried out using spinner flask.The results showed that the cell culture solution had a TCID₅₀ value of 10^7.0/0.1 mL,the EID₅₀ was 10^7.5/0.1 mL,and the HA titer was 1∶256.Therefore,the application of the above conditions to culture NDV LaSota strain in BHK-21 cells was fully applicable to the large-scale production of the NDV,and in line with the current veterinary biological products and inspection manufacturing regulations.

Key words: Newcastle disease virus (NDV); LaSota strain; BHK-21 cell; proliferation

CLC Number:

S858.31

DONG Bingmei, SUN Peijiao, MIAO Lizhong, SHEN Zhiqiang, HAN Wenyu. Optimization About Proliferated Condition of Newcastle Disease Virus LaSota Strain in BHK-21 Cells[J]. , 2019, 46(3): 858-864.

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Optimization About Proliferated Condition of Newcastle Disease Virus LaSota Strain in BHK-21 Cells

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