Establishment And Preliminary Application Of A Stable Vero Cell Line With Knocked-down P53 Gene Expression

p53 gene exists in a wide variety of animal cells, p53 gene has a similar gene structure in different species of animals. p53 protein encoded by p53 gene is called tumor suppressor protein, which may involve cell cycle regulation, induction of apoptosis, involved in cell differentiation and aging, anti-virus and other biological functions. When cells are subject to various external stress such as radiation, hypoxia, pathogen infection, as a transcription factor, p53 will be activated, so that it will lead to the expression of downstream genes, resulting in a series of biological effects, which aim at protecting genomes from external damage, so p53 is known as the "guardian of the genome."The biological function of p53 is so important that cell biologists and medical scientists are interested in this fields. To study the role of p53 and its mediated signal transduction pathway during animal virus infection, this study first according to pre-screening an effective small interfering RNA aiming at p53 gene fragment, using RNA interference technology to construct the p53 gene RNA interference lentiviral expression vector, and a three-plasmid lentivirus expression system package the recombinant lentivirus, at last, viral titer is determined in 293T cells.Vero cells are infected by pre-packaged recombinant lentivirus, and positive cells are screensd by puromycin. To purify positive cell lines, single cell clonies are obtained by partial digestion. p53 expression are detected by using RT-PCR and Western blot and p53 transcription activation function are analyzed by using luciferase reporting system. The results show that: compared with control cells, lentivirus-mediated shRNA effectively silence the expression of p53 gene and transcriptional activity of p53 is significantly decreased. Therefore, a stable Vero cell line with knocked-down p53 gene expression is obtained in vitro.Previously silencing expression of p53 gene in Vero cell line as a model, the animal herpesvirus PRV infect this cell, at different time points of virus infection, virus samples and whole cell protein samples are collected. The virus titer of different time point are measured by TCID50 method and multi-step growth curve of PRV is obtained; the transcription level changes of viral replication gene are determined by semi-quantitative RT-PCR; translation level changes are determined by Western blot. Compared the p53 knockdown-Vero cells with negative control group cells, these three indicators differences show that: p53-knockdown Vero cells compared with the negative control cells, the virus titer of PRV is decreased and virus replication gene mRNA expression and protein levels are also decreased, especially in virus infection later period, this trend is particularly evident. So this indicate that: p53 may be play role in late PRV infected host cells, this study made theoretical basis for studying between PRV and p53-mediated signal transduction pathway.In short, this is the first time to establish a stable p53 knockdown Vero cell line using lentiviral vector-mediated RNA interference , and this cell line is applied in studying animal virus replication . The Vero cell line will provide an enabling tool for studying the biological function of p53, and may be clarify p53 may play some potential role during some animal virus infection, so as to provide a theoretical basis for studying new anti-viral drugs.