title: Construction of eukaryotic expression vector with mBD1-mBD3 fusion genes and exploring its activity against influenza A virus.
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 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.
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. hxliwanyi@126.com.
authors.forename: Wanyi
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. yanfeng_1983@126.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. kuangyu20001113@163.com.
authors.forename: Yu
authors.initials: Y
authors.lastname: Kuang
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. h_zwcn@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zeng
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. yangyuan1114@163.com.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: West China Second University Hospital, Sichuan University, Chengdu 610041, China. lihong09@163.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. jjzz1879@163.com.
authors.forename: Zhonghua
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China. lmy3985@sina.com.
authors.forename: Mingyuan
authors.initials: M
authors.lastname: Li
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000008:administration & dosage*; D001688:Biological Products / Q000008:administration & dosage*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology*; D004195:Disease Models, Animal; D004285:Dogs; D005260:Female; D005822:Genetic Vectors; D009980:Influenza A virus / Q000276:immunology*; D008168:Lung / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D009976:Orthomyxoviridae Infections / Q000517:prevention & control*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D016019:Survival Analysis; D014157:Transcription Factors / Q000235:genetics / Q000276:immunology*; D019562:Viral Load; D014776:Virus Cultivation
chemical_list: D000998:Antiviral Agents; D001688:Biological Products; D004268:DNA-Binding Proteins; C489323:Mbd1 protein, mouse; C492013:Mbd3 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors