Generation of recombinant Bacillus subtilis expressing Porcine Epidemic Diarrhea Virus (PEDV) S1 protein in vegetative cell

  • Abstract
  • Keywords
  • References
  • PDF
  • Abstract

    Porcine epidemic diarrhea virus (PEDV) is a mucosal (gut surface) pathogen that causes severe diarrhea in piglets; thus, a vaccine capable of inducing gut-mucosal immune response is crucial for controlling PEDV infection. Bacillus subtilis has been considered a choice for vaccine delivery to the gut mucosa. In this study, we aimed to generate recombinant B. subtilis that can produce PEDV S1 protein in vegetative cell. Two promoters, PrrnO and PgsiB-PsecA, were selected for an early and high yield expression of PEDV S1 gene in B. subtilis vegetative cell and germinating spore. Promoters, PrrnO and PgsiB-PsecA, were linked to the 5’ end of the fusion gene pgsA-PEDVS1 and the fusion genes were then inserted into plasmid pDG1662. Recombinant B. subtilis strains were generated by integrating the fusion genes into B. subtilis 168 chromosome via double crossover homologous recombination. PCR amplification and amylase activity assay confirmed integration of the fusion genes into B. subtilis chromosome at amyE locus. Expression of the pgsA-PEDVS1 in B. subtilis vegetative cells germinating from spores was then studied at 2, 4, 8 and 16 hours of culture. Tested by western blot analysis, although only cleaved products of PgsA-PEDVS1 protein were observed, expression levels of pgsA-PEDVS1 under the control of both promoters were comparable at all time points. Importantly, PgsA-PEDVS1 protein could be detected as early as 2 hours after spore inoculation in LB medium. This study suggests that both PgsiB-PsecA and PrrnO promoters can be used for efficient production of PEDV S1 in germinating spore and vegetative cell and may be applicable for expression of other heterologous genes in B. subtilis vegetative cell.



  • Keywords

    Bacillus Subtilis, PEDV, Spike Protein, Mucosal Vaccine

  • References

      [1] Jing B, et al. (2012), "Complete genome sequence of porcine epidemic diarrhea virus strain AJ1102 isolated from a suckling piglet with acute diarrhea in China". Journal of Vorology 86(19), 10910-10911.

      [2] Suo S, et al. (2012), "Immune responses induced by DNA vaccines bearing Spike gene of PEDV combined with porcine IL-18." Virus Research 167, 259-266.

      [3] Chen J, et al. (2013), "Detection and molecular diversity of spike gene of porcine epidemic diarrhea virus in China". Viruses 5, 2601-2613.

      [4] Lin CM, et al. (2015), "Experimental infection of a US spike-insertion deletion porcine epidemic diarrhea virus in conventional nursing piglets and cross-protection to the original US PEDV infection". Veterinary research 46, 134.

      [5] Kwonil J, et al. (2015), "Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunprophylaxis". The Veterinary Journal 15, 1-35.

      [6] Teva P, et al. (2015), "Synergism of regulatory elements in sigma and sigmaB dependent promoters enhances recombinant protein expression in Bacillus subtilis." Journal of Bioscience and Bioengineering 120(4), 470-475.

      [7] Casula G, et al. (2002), " Bacillus probiotics: spore germination in the gastrointestine tract". Applied and environmental, microbiology 68(5), 2344-2352.

      [8] Stephen T, et al. (2008), "Bacillus subtilis spore germinate in the chicken gastrointestinal tract". Applied and Environmental Microbiology 74(16), 5254-5258.

      [9] Renata DS, et al. (2014), "Gut adhesive Bacillus subtilis spore as a platform for mucosal delivery of antigen." Infection and Immunity 82, 1414-1423.

      [10] Leser DT, et al. (2007), "Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pig". Journal of applied microbiology 104, 1025-1033.

      [11] Ashiuchi M, et al. (2001), "Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis". Eur. J. Biochem 268, 5321-5328.

      [12] Samarrai W, et al. (2011), "Differential responses of Bacillus subtilis rRNA promoters to nutritional stress". Journal of bacteriology 193(3), 723-733.

      Phanaksri T, et al. (2015), "Synergism of regulatory elements in sigma-B and sigma-A dependent promoters enhances recombinant protein expression in Bacillus subtilis". Journal of bioscience and bioengineering 120(4), 470-475




Article ID: 10953
DOI: 10.14419/ijet.v7i2.10.10953

Copyright © 2012-2015 Science Publishing Corporation Inc. All rights reserved.