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




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1

Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China

2

West China Second University Hospital, Sichuan University, Chengdu 610041, China

3

State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, China



These authors contributed equally to this work.





*

Author to whom correspondence should be addressed.



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. View Full-Text

Keywords: influenza A virus; mouse beta-defensin; pcDNA3.1+-mBD1-mBD3; overlap-PCR; anti-virus activity influenza A virus; mouse beta-defensin; pcDNA3.1+-mBD1-mBD3; overlap-PCR; anti-virus activity





Autor: Wanyi Li 1,†, Yan Feng 1,†, Yu Kuang 1, Wei Zeng 1, Yuan Yang 1, Hong Li 2, Zhonghua Jiang 1 and Mingyuan Li 1,3,*

Fuente: http://mdpi.com/



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