Viruses 2014, 6(3), 1237-1252; doi:10.3390/v6031237
Article

Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus

1,†email, 1,†email, 1email, 1email, 1email, 2email, 1email and 1,3,* email
Received: 29 November 2013; in revised form: 11 February 2014 / Accepted: 24 February 2014 / Published: 13 March 2014
(This article belongs to the Section Antivirals & Vaccines)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract: 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 TCID50 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.
Keywords: influenza A virus; mouse beta-defensin; pcDNA3.1(+)/mBD1-mBD3; overlap-PCR; anti-virus activity
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MDPI and ACS Style

Li, W.; Feng, Y.; Kuang, Y.; Zeng, W.; Yang, Y.; Li, H.; Jiang, Z.; Li, M. Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus. Viruses 2014, 6, 1237-1252.

AMA Style

Li W, Feng Y, Kuang Y, Zeng W, Yang Y, Li H, Jiang Z, Li M. Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus. Viruses. 2014; 6(3):1237-1252.

Chicago/Turabian Style

Li, Wanyi; Feng, Yan; Kuang, Yu; Zeng, Wei; Yang, Yuan; Li, Hong; Jiang, Zhonghua; Li, Mingyuan. 2014. "Construction of Eukaryotic Expression Vector with mBD1-mBD3 Fusion Genes and Exploring Its Activity against Influenza A Virus." Viruses 6, no. 3: 1237-1252.


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