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:
      目的 构建稳定过表达波形蛋白(Vimentin,VIM)基因的鸡肝癌(Leghorn male hepatoma,LMH)细胞系以提高鹅星状病毒(Goose astrovirus,GAstV)增殖效率。
      方法 从鹅源细胞中通过PCR扩增VIM基因,结合同源重组的方法将其连接至慢病毒基因过表达系统载体pLV-sfGFP (2A) Puro,获得重组慢病毒质粒pLV-sfGFP (2A) Puro-VIM-Flag(pLV-VIM);利用293T细胞包装重组慢病毒颗粒,随后感染LMH细胞,并利用嘌呤霉素进行筛选以获得目标细胞;通过Western blot、RT-qPCR、间接免疫荧光试验和TCID50测定等方法评估VIM对GAstV增殖效率的影响;最后通过抗体阻断试验分析细胞表面VIM对GAstV侵入的影响。
      结果 经测序和酶切鉴定,成功构建了重组慢病毒载体pLV-VIM;经筛选后获得了过表达VIM基因的LMH细胞系(LMH-VIM)及其对照组细胞系(LMH-NC)。Western blot结果显示,LMH-VIM细胞中波形蛋白的表达水平显著高于LMH-NC。接种病毒后发现,过表达VIM显著上调LMH-VIM细胞中GAstV的mRNA和蛋白表达水平,以及细胞上清液中的病毒滴度。抗体阻断试验表明,细胞表面VIM可促进GAstV侵入细胞。
      结论 本研究为提高GAstV在体外细胞培养的增殖滴度提供了新的见解和思路,对GAstV感染引起的雏鹅痛风疾病的疫苗研发、生产和该疾病的防控具有重要意义。

       

      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.

       

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