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.
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。
