Chinese Journal of Oceanology and Limnology

, Volume 28, Issue 6, pp 1254–1260 | Cite as

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

  • Ye Yuan (袁野)
  • Xiuli Wang (王秀利)
  • Sheping Guo (郭设平)
  • Yang Liu (刘洋)
  • Hui Ge (葛辉)
  • Xuemei Qiu (仇雪梅)
Biology

Abstract

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene (flaA) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can be used for further functional and structural studies.

Keyword

Vibrio parahaemolyticus flagellin subunit gene (flaAcloning prokaryotic expression characterization 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Arora S K, Ritchings B W, Almira E C, Lory S, Ramphal R. 1997. A transcriptional activator, FleQ, regulates mucin adhesion and flagellar gene expression in Pseudomonas aeruginosa in a cascade manner. J. Bacteriol., 179(17): 5 574–5 581.Google Scholar
  2. Brandfass C, Karlovsky P. 2008. Upscaled CTAB-based DNA extraction and real-time PCR assays for Fusarium culmorum and F. graminearum DNA in plant material with reduced sampling error. Int. J. Mol. Sci., 9(11): 2 306–2 321.CrossRefGoogle Scholar
  3. Chávez A, García-Huante Y, Ruiz B, Langley E, Rodríguez-Sanoja R. Sanchez S. 2009. Cloning and expression of the sco2127 gene from Streptomyces coelicolor M145. J. Ind. Microbiol. Biotechnol., 36(5): 649–654.CrossRefGoogle Scholar
  4. Das M, Chopra A K, Wood T, Peterson J W. 1998. Cloning, sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum. FEMS Microbiol. Lett., 165(2): 239–246.Google Scholar
  5. Datta S, Janes M E, Simonson J G. 2008. Immunomagnetic separation and coagglutination of Vibrio parahaemolyticus with anti-flagellar protein monoclonal antibody. Clin. Vaccine. Immunol., 15(10): 1 541–1 546.CrossRefGoogle Scholar
  6. Fan J F, Song L C, Wang B, Zang H M, Liang Y B. 2006. Preliminary study on one pathogenic bacterium-Vibrio parahaemolyticus associated with red body disease in Litopenaeus vannamei. Marine Sciences, 30(4): 40–44. (in Chinese with English abstract)Google Scholar
  7. Jacchieri S G, Torquato R, Brentani R R. 2003. Structural study of binding of flagellin by Toll-like receptor 5. J. Bacteriol., 185(14): 4 243–4 247.CrossRefGoogle Scholar
  8. Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 16(2): 111–120.CrossRefGoogle Scholar
  9. Klose K E, Mekalanos J J. 1998. Differential regulation of multiple flagellins in Vibrio cholerae. J. Bacteriol., 180(2): 303–316.Google Scholar
  10. Lee J H, Rho J B, Park K J, Kim C B, Han Y S, Choi S H, Lee K H, Park S J. 2004. Role of flagellum and motility in pathogenesis of Vibrio vulnificus. Infect. Immun., 72(8): 4 905–4 910.CrossRefGoogle Scholar
  11. Lee K K, Liu P C, Huang C Y. 2003. Vibrio parahaemolyticus infectious for both humans and edible mollusk abalone. Microbes. Infect, 5(6): 481–485.CrossRefGoogle Scholar
  12. Liu Z, Miyashiro T, Tsou A, Hsiao A, Goulian M, Zhu J. 2008. Mucosal penetration primes Vibrio cholerae for host colonization by repressing quorum sensing. Proc. Natl. Acad. Sci. USA., 105(28): 9 769–9 774.Google Scholar
  13. Macnab R M. 2003. How bacteria assemble flagella. Annu. Rev. Microbiol., 57: 77–100.CrossRefGoogle Scholar
  14. McCarter L L. 2001. Polar flagellar motility of the Vibrionaceae. Microbiol. Mol. Biol. Rev., 65(3): 445–462.CrossRefGoogle Scholar
  15. McGee K, Hörstedt P, Milton D L. 1996. Identification and characterization of additional flagellin genes from Vibrio anguillarum. J Bacteriol., 178(17): 5 188–5 198.Google Scholar
  16. Millikan D S, Ruby E G. 2004. Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization. J. Bacteriol., 186(13): 4 315–4 325.CrossRefGoogle Scholar
  17. Milton D L, O’Toole R, Hörstedt P, Wolf-Watz H. 1996. Flagellin A is essential for the virulence of Vibrio anguillarum. J. Bacteriol., 178(5): 1 310–1 319.Google Scholar
  18. Moorthy S, Watnick P I. 2004. Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization. J. Bacteriol., 186(13): 4 315–4 325.Google Scholar
  19. Ormonde P, Hörstedt P, O’Toole R, Milton D L. 2000. Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum. J. Bacteriol., 182(2): 2 326–2 328.Google Scholar
  20. Qian R, Xiao Z, Zhang C, Chu W, Mao Z, Yu L. 2008. Expression and purification of two major outer membrane proteins from Vibrio alginolyticus. World. J. Microbiol. Biotechnol., 24: 245–251.CrossRefGoogle Scholar
  21. Qin Y X, Wang J, Wang S F, Su Y Q, Zhuang X. 2007. Study on the expression of recombined plasmid pcFlaA in Epinephelus awoara. High Tech. Lett., 17(7): 761–764. (in Chinese with English abstract)Google Scholar
  22. Ramos H C, Rumbo M, Sirard J C. 2004. Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa. Trends Microbiol., 12(11): 509–517.CrossRefGoogle Scholar
  23. Saitou N, Nei M. 1987. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4(4): 406–425.Google Scholar
  24. Shapiro L. 1995. The bacterial flagellum: from genetic net-work to complex architecture. Cell, 80(4): 525–527.CrossRefGoogle Scholar
  25. Thompson F L, Iida T, Swings J. 2004. Biodiversity of vibrios. Microbiol. Mol. Biol. Rev., 68(3): 403–431.CrossRefGoogle Scholar
  26. Thompson J D, Higgins D G, Gibson T J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res., 22(22): 4 673–4 680.CrossRefGoogle Scholar
  27. Wu Z L, Nie J, Bao H N. 2001. Expression of thermostable direct hemolysin gene of Vibrio parahaemolyticus in E. coli and activity of expressed product. J. Cell. Mol. Immunol., 17(5): 426–427.Google Scholar
  28. Young G M, Smith M J, Minnich S A, Miller V L. 1999. The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility. J. Bacteriol., 181(9): 2 823–2 833.Google Scholar
  29. Zhuang X, Qin Y X, Su Y Q, Wang J, Ding S X. 2007. Cloning and sequencing of flaA gene of Vibrio harveyi and construction of its eukaryotic expression recombinant plasmid. Acta Oceanol. Sin., 29(6): 74–79. (in Chinese with English abstract)Google Scholar
  30. Zorrilla I, Arijo S, Chabrillon M, Diaz P, Martinez-Manzanares E, Balebona M C, Moriñigo M A. 2003. Vibrio species isolated from diseased farmed sole, Solea senegalensis (Kaup), and evaluation of the potential virulence role of their extracellular products. J. Fish. Dis., 26(2): 103–108.CrossRefGoogle Scholar

Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer Berlin Heidelberg 2010

Authors and Affiliations

  • Ye Yuan (袁野)
    • 1
  • Xiuli Wang (王秀利)
    • 1
  • Sheping Guo (郭设平)
    • 1
  • Yang Liu (刘洋)
    • 1
  • Hui Ge (葛辉)
    • 1
  • Xuemei Qiu (仇雪梅)
    • 1
  1. 1.College of Life Sciences and BiotechnologyDalian Fisheries UniversityDalianChina

Personalised recommendations