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

doi:10.11843/j.issn.0366-6964.2021.02.016

Abstract

Abstract: 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.

Key words: TIGAR, apoptosis, anti-apoptosis, Newcastle disease virus, virus titer

CLC Number:

S852.659.5

LI Yonghua, LIU Wei, XU Zhikai, LIU Weiwei, SUN Yingjie, QIU Xusheng, TAN Lei, SONG Cuiping, LIAO Ying, DING Chan, MENG Chunchun. Construction of BHK-21 Cell Line Stably Expressing TIGAR Gene and Evaluation of Its Proliferation Effect on Newcastle Disease Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(2): 440-449.

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

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