Abstract:
Objective To improve the proliferation efficiency of goose astrovirus (GAstV) by constructing a leghorn male hepatoma (LMH) cell line stably overexpressing vimentin (VIM) gene.
Method The VIM gene was amplified from goose-derived cells by PCR and subsequently ligated into the lentiviral gene overexpression system vector pLV-sfGFP (2A) Puro by homologous recombination to obtain the recombinant lentiviral plasmid pLV-sfGFP (2A) Puro-VIM-Flag(pLV-VIM). The 293T cells were used to package the recombinant lentivirus particles, which subsequently infected LMH cells and were screened with puromycin to obtain target cells. The effect of VIM on GAstV proliferation efficiency was evaluated by Western blot, RT-qPCR, indirect immunofluorescence and TCID50 assays. Finally, the effect of VIM on GAstV invasion was analyzed by antibody blocking test.
Result The recombinant lentiviral vector pLV-VIM was successfully constructed as confirmed by sequencing and restriction enzyme digestion. After screening, LMH-VIM cell line overexpressing the VIM gene (LMH-VIM) and its control group cell line (LMH-NC) were obtained. The results of Western blot showed that the expression level of VIM protein in LMH-VIM cells was significantly higher than that in LMH-NC cells. Overexpression of VIM significantly upregulated GAstV mRNA and protein expression levels in LMH-VIM cells, as well as viral titer in the cell supernatant. Antibody blocking test showed that cell surface VIM could actively promote GAstV invasion into cells.
Conclusion The study provides a new insight and ideas for improving the proliferation titer of GAstV in the in-vitro cell culture and is of great significance for the vaccine research and development, as well as prevention and control of gosling gout disease caused by GAstV infection.
下载: