鸡新城疫病毒LaSota株在BHK-21细胞上增殖条件的优化

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

本研究旨在对鸡新城疫病毒(Newcastle disease virus,NDV)LaSota株在BHK-21细胞上的增殖条件进行优化,为实现应用细胞系生产NDV提供参考。在确定BHK-21细胞TPCK-胰酶耐受浓度的基础上,在培养基中添加不同浓度的TPCK-胰酶、新生牛血清及调节不同pH,通过对细胞培养液细胞半数感染量(TCID_(50))的测定,确定最适胰酶浓度、血清浓度及pH;通过固定接毒量而添加不同数量细胞或固定细胞数量而接种不同量的病毒,确定最适细胞量及接毒量;将BHK-21细胞接种NDV LaSota株后,通过测定不同时间细胞上清液TCID_(50)绘制增殖曲线,确定最佳收毒时间;应用转瓶对该病毒进行扩大培养,并进行TCID_(50)、鸡胚半数感染量(EID_(50))与血清凝集价(HA)的测定。结果显示,NDV LaSota株在BHK-21细胞上增殖的最适TPCK-胰酶浓度为3μg/mL,最适接毒量为10^(6.375) EID_(50),细胞数量为1×10~6个,培养基pH为7.2,且可用无血清培养基进行培养,BHK-21细胞接种NDV LaSota株后最佳收毒时间为34～36 h。应用BHK-21细胞采用转瓶培养的方式对NDV LaSota株进行增殖,结果显示,细胞培养液TCID_(50)值为10^(7.0)/0.1 mL,EID_(50)为10^(7.5)/0.1 mL,HA效价为1∶256。因此,应用以上条件增殖培养NDV LaSota株,完全适用于该病毒的规模化生产,且符合现行的兽医生物制品与检验制造规程。

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 10^6.375EID50,the number of cells was 1×10^6,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 EID 50 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.