Construction Of BHK-21 Cell Line Stably Expressing TIGAR Gene And Evaluation Of Its Anti-apoptotic Effect

Downstream signaling of p53 regulates the TP53-induced glycolysis and apoptosis regulator（TIGAR）,which can regulate the glycolysis level and have anti-apoptotic function.As cell death caused induced Newcastle disease virus is belong to apoptosis,increasing the anti-apoptotic level of host cells it may helpful to prolong the survival time of cells and then increase the titers of virus.This study aims to prolong the cell survival time after virus infection by constructing a cell line with over-expression of TIGAR gene in order to pave the way for the production of Newcastle disease virus vaccine by cell culture method.1.Construction of chicken TIGAR gene eukaryotic expression plasmid and evaluation of its anti-apoptotic functionPrimers were designed according to the predicted sequence of TIGAR gene published in GenBank（accession number:XM₄17232.6）and the gene amplified from the SPF chicken spleen by RT-PCR,then PCR products were cloned into expression vector（Flag-CMV14）and verified by DNA sequencing.Homology analysis was performed by constructing the phylogenetic tree of chicken TIGAR genes and compared with mammals and aquatic animals TIGAR gene.The recombinant plasmid was transfected into DF1 cells,and infected with Newcastle disease virus to induce apoptosis.Western blot was performed to determine the expression of TIGAR gene and the cleavage level of PARP protein.At the same time the recombinant plasmid（Flag-TIGAR）was transfected into DF1 cells and incubated with apoptosis inducer staurosporine for 2 hours before sample collection,then cells were collected at 24 h and 48 h after transfection,and evaluated the apoptosis level by flow cytometry.The TIGAR gene was amplified by RT-PCR,and a band appeared at 843 bp,which was consistent with the prediction.The constructed TIGAR eukaryotic expression plasmid（Flag-TIGAR）was sequenced,and the results showed that the sequence of amplified TIGAR gene were almost consistent with the predicted sequence which published on GenBank.Western blot results show:cleavaged PARP bands were existed,and the expression level in transfected recombinant plasmid（Flag-TIGAR）group was significantly different from non-transfected plasmid（MOCK）group and transfected empty vector（Flag-CMV14）group.Flow cytometry results showed that the total apoptotic rate of transfection with Flag-CMV14 for 24 h and 48 h was higher than transfection Flag-CMV14.2.Construction of BHK-21 cell line stably expression TIGAR gene and detection of its anti-apoptosis functionAccording to the predicted TIGAR gene published in GenBank（accession number:XM₀05084096.3）,primers were designed,and amplified the TIGAR gene from BHK-21 cells by RT-PCR.Amplified products were cloned into expression vector（PMD-19T）and positive clone was verified by DNA sequencing.Construction of lentivirus packaging plasmid phage-TIGAR,using chicken TIGAR gene and hamster TIGAR gene to construct stable expression cell lines（BHK-21-chTIGAR and BHK-21-hamTIGAR）using lentivirus packaging system.The stability of the cell line was detected by Western blot and qPCR,the natural apoptosis rate of the cell line was measured by flow counting,and the growth curve of Newcastle disease virus on the constructed cell line was plotted.The results showed that the amplified TIGAR sequence of hamster was consistent with the predicted gene sequence available on the GenBank.The constructed BHK-21-chTIGAR cells and BHK-21-hamTIGAR cells were identified by Western blot,DNA sequencing and indirect immunofluorescence assay,indicating establishment of the constructed cell lines and stable expression of TIGAR gene expression.The results of the stability of the cell line of the Western blot and the qPCR show that the TIGAR gene can be stably expressed in the BHK-21 cell.The results of the detection of the cell’s natural apoptosis rate show that the BHK-21-chTIGAR is higher than wild-type BHK-21 cell,on the contrary the natural apoptosis rate of the BHK-21-hamTIGAR cell is lower than wild-type BHK-21 cell.After NDV induced apoptosis,the apoptosis rate of BHK-21-hamTIGAR cells was lower than wild type BHK-21 cells and BHK-21-chTIGAR cells.The results of virus growth curve showed that the TCID50 of BHK-21-hamTIGAR cells was higher than BHK-21-chTIGAR cells and wild type BHK-21 cells.In conclusion,the BHK-21-hamTIGAR cell line has obvious anti-apoptotic function and it can support high-level replication of Newcastle disease virus,which is expected to be further developed as a cell line for produce of Newcastle disease virus vaccine.