构建 mBD1-mBD3 融合基因真核表达载体并探索其抗甲型流感病毒活性。
Construction of eukaryotic expression vector with mBD1-mBD3 fusion genes and exploring its activity against influenza A virus.
机构信息
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
West China Second University Hospital, Sichuan University, Chengdu 610041, China.
出版信息
Viruses. 2014 Mar 13;6(3):1237-52. doi: 10.3390/v6031237.
Influenza (flu) pandemics have exhibited a great threat to human health throughout history. With the emergence of drug-resistant strains of influenza A virus (IAV), it is necessary to look for new agents for treatment and transmission prevention of the flu. Defensins are small (2-6 kDa) cationic peptides known for their broad-spectrum antimicrobial activity. Beta-defensins (β-defensins) are mainly produced by barrier epithelial cells and play an important role in attacking microbe invasion by epithelium. In this study, we focused on the anti-influenza A virus activity of mouse β-defensin 1 (mBD1) and β defensin-3 (mBD3) by synthesizing their fusion peptide with standard recombinant methods. The eukaryotic expression vectors pcDNA3.1(+)/mBD1-mBD3 were constructed successfully by overlap-PCR and transfected into Madin-Darby canine kidney (MDCK) cells. The MDCK cells transfected by pcDNA3.1(+)/mBD1-mBD3 were obtained by G₄₁₈ screening, and the mBD1-mBD3 stable expression pattern was confirmed in MDCK cells by RT-PCR and immunofluorescence assay. The acquired stable transfected MDCK cells were infected with IAV (A/PR/8/34, H1N1, 0.1 MOI) subsequently and the virus titers in cell culture supernatants were analyzed by TCID5₅₀ 72 h later. The TCID₅₀ titer of the experimental group was clearly lower than that of the control group (p < 0.001). Furthermore, BALB/C mice were injected with liposome-encapsulated pcDNA3.1(+)/mBD1-mBD3 through muscle and then challenged with the A/PR/8/34 virus. Results showed the survival rate of 100% and lung index inhibitory rate of 32.6% in pcDNA3.1(+)/mBD1-mBD3group; the TCID₅₀ titer of lung homogenates was clearly lower than that of the control group (p < 0.001). This study demonstrates that mBD1-mBD3 expressed by the recombinant plasmid pcDNA3.1(+)/mBD1-mBD3 could inhibit influenza A virus replication both in vitro and in vivo. These observations suggested that the recombinant mBD1-mBD3 might be developed into an agent for influenza prevention and treatment.
流感（flu）大流行在历史上一直对人类健康构成巨大威胁。随着流感 A 病毒（IAV）耐药株的出现，有必要寻找新的药物来治疗和预防流感。防御素是一种具有广谱抗菌活性的小（2-6 kDa）阳离子肽。β-防御素（β-defensins）主要由屏障上皮细胞产生，在抵御上皮细胞中微生物入侵方面发挥着重要作用。在这项研究中，我们通过使用标准重组方法合成其融合肽，重点研究了小鼠β-防御素 1（mBD1）和β防御素-3（mBD3）抗流感 A 病毒的活性。通过重叠 PCR 成功构建了真核表达载体 pcDNA3.1(+)/mBD1-mBD3，并转染到 Madin-Darby 犬肾（MDCK）细胞中。通过 G₄₁₈ 筛选获得转染 pcDNA3.1(+)/mBD1-mBD3 的 MDCK 细胞，并通过 RT-PCR 和免疫荧光检测证实 mBD1-mBD3 在 MDCK 细胞中的稳定表达模式。随后用 IAV（A/PR/8/34，H1N1，0.1 MOI）感染获得的稳定转染的 MDCK 细胞，72 小时后通过 TCID5₅₀ 分析细胞培养上清液中的病毒滴度。实验组的 TCID5₅₀ 滴度明显低于对照组（p < 0.001）。此外，BALB/C 小鼠通过肌肉注射包裹在脂质体中的 pcDNA3.1(+)/mBD1-mBD3，然后用 A/PR/8/34 病毒进行攻毒。结果显示，pcDNA3.1(+)/mBD1-mBD3 组的存活率为 100%，肺指数抑制率为 32.6%；肺匀浆的 TCID5₅₀ 滴度明显低于对照组（p < 0.001）。本研究表明，重组质粒 pcDNA3.1(+)/mBD1-mBD3 表达的 mBD1-mBD3 可在体外和体内抑制流感 A 病毒的复制。这些观察结果表明，重组 mBD1-mBD3 可能被开发为一种流感预防和治疗药物。
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