Construction Of BHK-21 Cell Line Stably Overexpression Chicken ATG12 And ATG16L1 Gene And The Proliferation Of Newcastle Disease Virus

Newcastle disease?ND?is an acute and highly contagious disease caused by the Newcastle disease virus?NDV?,which causes serious economic loss to the poultry industry.The main virulent strain of the chicken group in our country is the NDV of gene VII.In view of the prevention and control of ND,our country mainly takes the vaccination.The main production method of the vaccine is chicken embryo culture.Compared with the chicken embryo,the use of cells as culture matrix has the advantages of easy operation and more controllable.This study is aimed at the prevention and control of NDV from the aspect of virus production.In this study,lentivirus vector pHBLV-CMV-MCS-EF1-ZSgreen-puro was used to construct BHK-21 cell lines that overexpressed chicken ATG12 and ATG16L1 genes.The positive cells screened by puromycin were named BHK-21-ATG12 cells and BHK-21-ATG16L1 cells.Under the fluorescence microscope,the green fluorescence of BHK-21-ATG12 cells and BHK-21-ATG16L1 cells was rich,and the positive rate of BHK-21-ATG12 cells and BHK-21-ATG16L1 cells was more than 90%by flow cytometry;qRT-PCR detected that the relative expression of ATG12 and ATG16L1 in BHK-21-cells and cells was about 420 times and 280 times of that of parental cells.BHK-21-ATG12 cells and BHK-21-ATG16L1 cell proteins were detected by Westem Blot,and specific bands were detected at about 38.6 kD and 93.6 kD,which were consistent with the expected size.After 15 successive generations of cells,there was no difference between the positive cells and the parent cells under the optical microscope and the relative expression of ATG12 and ATG16L1 on the mRNA level in BHK-21-ATG12 cells and BHK-21-ATG16L1 cells by qRT-PCR was about 380 times and 260 times that of the parental cells.The results indicate that ATG12 and ATG16L1 have been overexpressed in the constructed BHK-21 cell line and have genetic stability.Then the proliferation of Newcastle disease rGM-VII m strain in these 2 recombinant cells was detected.The rGM-VII m virus was inoculated into BHK-21-ATG12 cells,BHK-21-ATG16L1 cells and BHK-21 cells.Cell and cell supernatants were collected after12,24,36,48,72,96,120,144 and 168 h respectively.The cell samples were extracted with RNA and qRT-PCR to detect the virus replication in the cells.The titers of viral HA and TCID₅₀ in the supernatant samples were measured.The results showed that the relative expression of NP gene in rGM-VII m in BHK-21-ATG12 cells was significantly higher than that of parental cells in 48 h after poisoningt?P<0.01?,and the relative expression of NP gene in rGM-VII m in BHK-21-ATG16L1 cells was significantly higher than that of parental cells in 48 h after poisoningt?P<0.001?.After 48 h,BHK-21-ATG12 cell supernatant HA and TCID₅₀ were 2.46 times?P<0.05?and 15.49 times?P<0.001?respectively.BHK-21-ATG16L1 cells supernatant HA,TCID₅₀ were 3.25 times as much as parental cells?P<0.01?and 131.83 times?0.001?.Compared with BHK-21 cells,the proliferation of rGM-VII m virus on BHK-21-ATG12 cells and BHK-21-ATG16L1 cells was higher at 48 h after infection.In this study,we successfully constructed BHK-21-ATG12 cells and BHK-21-ATG16L1 cells that stably overexpressed chicken ATG12 and ATG16L1 genes.Compared with BHK-21 cells,rGM-VII m virus has a higher proliferation titer on BHK-21-ATG12cells and BHK-21-ATG16L1 cells,indicating that it is more suitable for the proliferation of rGM-VII virus and has the potential to produce rGM-VII m virus vaccine.