稳定表达TIGAR基因的BHK-21细胞系的构建及其对新城疫病毒增殖效果的评价

TP53诱导的糖酵解和凋亡调节因子（TP53-induced glycolysis and apoptosis regulator，TIGAR）是p53下游的靶基因，具有调节糖酵解水平和抗细胞凋亡功能。由于新城疫病毒（NDV）引起细胞死亡是通过诱导凋亡产生，所以提高宿主细胞的抗凋亡水平，有助于延长细胞存活时间进而提高子代病毒的滴度。本研究根据GenBank中预测仓鼠TIGAR基因和鸡TIGAR基因设计引物，分别扩增仓鼠和鸡的TIGAR基因，并构建各自的慢病毒包装质粒，以适合NDV增殖的BHK-21细胞为基础构建稳定过表达TIGAR细胞系（BHK-21-chTIGAR和BHK-21-hamTIGAR）。使用Western blot、DNA测序和qPCR对TIGAR基因表达水平进行鉴定，Western blot和流式仪检测细胞的自然凋亡率。选取新城疫病毒强中弱3种毒株分别感染构建的细胞系，测定病毒滴度。结果显示：重组细胞系BHK-21-chTIGAR细胞和BHK-21-hamTIGAR细胞经过Western blot和DNA测序等检测表明构建成功，TIGAR基因表达量高且无野生型BHK-21细胞污染。qPCR检测细胞系的稳定性结果显示，TIGAR基因在BHK-21细胞中经过30次传代均可稳定表达。Western blot和流式细胞检测结果显示：BHK-21-hamTIGAR细胞的凋亡率低于野生型BHK-21细胞和BHK-21-chTIGAR细胞；病毒生长曲线结果显示：强毒株ZJ1病毒感染BHK-21-hamTIGAR细胞后的TCID₅₀（5.67，72 h）高于BHK-21-chTIGAR细胞（5.53，72 h）和野生型BHK-21细胞（5.27，72 h）；中强毒株SH15病毒感染BHK-21-hamTIGAR细胞后的TCID₅₀（4.90，96 h）要高于BHK-21-chTIGAR细胞（4.57，96 h）和野生型BHK-21细胞（4.67，96 h）；弱毒株LaSota病毒感染BHK-21-hamTIGAR细胞后的TCID₅₀（4.07，96 h）要高于BHK-21-chTIGAR细胞（3.90，96 h）和野生型BHK-21细胞（3.77，96 h）。综上所述，所构建的BHK-21-hamTIGAR细胞系具有明显的抗凋亡功能，且有利于新城疫病毒的高水平复制，有望进一步开发完善成为新城疫病毒疫苗生产所用的细胞株。

Construction of BHK-21 Cell Line Stably Expressing TIGAR Gene and Evaluation of Its Proliferation Effect on Newcastle Disease Virus

TP53-induced glycolysis and apoptosis regulator,TIGAR is a target gene downstream of p53,which has the functions of regulating glycolysis and anti-apoptosis.Since the Newcastle disease virus(NDV)causes cell death by inducing apoptosis,increasing the anti-apoptotic level of host cells will prolong cell survival time and increase the titer of progeny viruses.In this study,primers were designed based on the predicted TIGAR gene of hamster and chicken on GenBank.Amplified the TIGAR gene to construct lentivirus packaging plasmid,then construct of the stable expression cell lines(BHK-21-chTIGAR and BHK-21-hamTIGAR)with helper plasmid.Western blot,DNA sequencing,and qPCR were used to identify cell lines.Western blot and flow cytometry was used to evaluate the apoptosis rate of cells.Three NDV strains with high,medium,and low pathogenicity,were selected to infect the constructed cell lines and to compare the virus titer with the wild-type BHK-21.The results of Western blot and DNA sequencing showed that the recombinant cell lines,BHK-21-chTIGAR cells and BHK-21-hamTIGAR cells,were successfully constructed and with high expression levels of the TIGAR gene without wild-type BHK-21 cells contamination.The stability of the cell lines detected by qPCR showed that the TIGAR gene can be continuously expressed after 30 times passages.The natural apoptosis rate of BHK-21-hamTIGAR cells was much lower than that of BHK-21-chTIGAR cells and wild-type BHK-21 cells.The virus growth curve demonstrated that no matter high,medium,and low pathogenicity of NDV strain all have higher virus titer in BHK-21-hamTIGAR cells compared to BHK-21-chTIGAR cells and wild-type BHK-21 cells.In conclusion,the BHK-21-hamTIGAR cell line has the obvious anti-apoptotic effect and is conducive to support the high-level replication of NDV.It has the potential to be further modified as a suitable cell line to produce NDV vaccines NDV for preparing vaccines.