Construction of LMH cell line overexpressing VIM gene and effect on replication efficiency of GAstV

TANG Zheng; XIANG Yong; SUN Minhua; FENG Saixiang; LIAO Ming; HUANG Yu; WAN Chunhe; LIU Rongchang; FU Guanghua; LUO Kaijian

doi:10.7671/j.issn.1001-411X.202407011

Journal of South China Agricultural University > 2025 > 46(3): 342-350. > DOI: 10.7671/j.issn.1001-411X.202407011

TANG Zheng, XIANG Yong, SUN Minhua, et al. Construction of LMH cell line overexpressing VIM gene and effect on replication efficiency of GAstV[J]. Journal of South China Agricultural University, 2025, 46(3): 342-350. DOI: 10.7671/j.issn.1001-411X.202407011

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Construction of LMH cell line overexpressing VIM gene and effect on replication efficiency of GAstV

1.
College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
2.
Institute of Animal Health, Guangdong Academy of Agricultural Sciences/Key Laboratory for Prevention and Control of Avian Influenza and Other Major Poultry Diseases, Ministry of Agriculture and Rural Affairs/Key Laboratory of Livestock Disease Prevention of Guangdong Province, Guangzhou 510640, China
3.
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences/Fujian Provincial Key Laboratory for Avian Diseases Control and Prevention, Fuzhou 350003, China

More Information

Received Date: July 09, 2024
Revised Date: September 10, 2024
Accepted Date: September 23, 2024
Available Online: February 26, 2025
Published Date: March 05, 2025

Graphical Abstract

Abstract

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.
- Vimentin gene,
- Leghorn male hepatoma cell line,
- Gene overexpression,
- Goose astrovirus,
- Proliferation efficiency

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References

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Construction of LMH cell line overexpressing VIM gene and effect on replication efficiency of GAstV

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