华南农业大学学报2025，Vol.46Issue(3)：342-350,9.DOI:10.7671/j.issn.1001-411X.202407011
过表达VIM基因的LMH细胞系构建及对GAstV增殖效率的影响
Construction of LMH cell line overexpressing VIM gene and effect on replication efficiency of GAstV
摘要
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.关键词
波形蛋白基因/鸡肝癌细胞系/基因过表达/鹅星状病毒/增殖效率Key words
Vimentin gene/Leghorn male hepatoma cell line/Gene overexpression/Goose astrovirus/Proliferation efficiency分类
畜牧业引用本文复制引用
唐铮,罗开健,向勇,孙敏华,冯赛祥,廖明,黄瑜,万春和,刘荣昌,傅光华..过表达VIM基因的LMH细胞系构建及对GAstV增殖效率的影响[J].华南农业大学学报,2025,46(3):342-350,9.基金项目
国家重点研发计划(2022YFD1801000) （2022YFD1801000）
"十四五"广东省农业科技创新十大主攻方向"揭榜挂帅"项目(2022SDZG02) （2022SDZG02）
福建省禽病防治重点实验室开放基金(FKADL-2024-01) （FKADL-2024-01）
