Colonization and Investigation of Vibrio Cholera Recombination Protein in E-Coli

 
 
 
  • Abstract
  • Keywords
  • References
  • PDF
  • Abstract


    Background and aim: Vibrio cholerae is a gram-negative bacterial pathogen that causes diarrheal disease. One of the most pathogenic factors of V. cholerae is toxin-coregulated pili. This pilus is required as the first factor in the colonization and bacterial persistence in the small intestine. Materials and Methods: In this study, V. cholerae toxin-coregulated pili A (TCPA) gene was amplified using PCR method. The above genes were purified and then expressed by being cloned into the pGEX4T-1 plasmid. Then the recombinant plasmid structure was introduced into the E. coli bacterium. Protein production was carried out by IPTG induction and optimization of culture conditions. The recombinant proteins were purified using Glutathione S-Transferase (GST) Assay Kit and western blot test was then carried out for confirmation of recombinant protein. Protein levels were measured using Bradford protein assay. Results: The results of the present study proved the successful expression of recombinant proteins in E. coli cells. The recombinant protein was purified by affinity chromatography. The reaction pattern between these proteins and their anti-antibodies showed that these proteins have antigenic properties. Conclusion: Since it was proved that these proteins have antigenic properties in this study, they may be used as an appropriate antigen for vaccination of V. cholera.

     

     


  • Keywords


    Polymerase chain reaction, Toxin-coregulated pili A gene, Vibrio Cholera.

  • References


      [1] Nguyen DT, Ngo TC, Tran HH, Le TH, Nguyen HT, Nguyen BM, et al. Characterization of Vibrio cholerae O139 of an Aquatic Isolate in Northern Vietnam. The open microbiology journal. 2012; .41:6

      [2] Janda J, Powers C, Bryant R, Abbott S. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clinical Microbiology Reviews. 1988; : 31, 245-.76

      [3] Butler SM, Camilli A. Going against the grain: chemotaxis and infection in Vibrio

      [4] Ehara M, Ishibashi M, Ichinose Y, Iwanaga M, Shimotori S, Naito T. Purification and partial characterization of fimbriae of Vibrio cholerae O1. Vaccine. 1987;5(4):283-8.

      [5] Taylor RK, Miller VL, Furlong DB, Mekalanos JJ. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proceedings of the National Academy of Sciences. 1987; 84(9): 2833-7.

      [6] Ramamurthy T, Yamasaki S, Takeda Y, Nair GB. Vibrio cholerae O139 Bengal: odyssey of a fortuitous variant. Microbes and infection. 2003; 5(4): 329-44.

      [7] Kaper JB, Glenn JR, and Levine MM. Cholera. Clin Microb. 1995;8(1):48–86.

      [8] Taylor RK, Kirn TJ, Bose N, Stonehouse E, Tripathi SA, Kováč P, et al. Progress towards development of a cholera subunit vaccine. Chemistry & biodiversity. 2004;1(7):1036-57.

      [9] Chen LY, Chen DY, Miaw J, Hu NT. XpsD, an outer sequence and comparison of predicated protein structural features to those of type 4 pilins. Infect. Immun.1996, 58:3042-9.

      [10] West PA, Colwell RR. Identification and classification of Vibrionaceae-an overview. Vibrios in the Environment. 1984:285-363.

      [11] Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science. 1996; 272(5270):1910-.4

      [12] Pal B, Khuntia H, Samal S, Kar S, Patnaik B. Epidemics of severe cholera caused by El Tor Vibrio cholerae O1 Ogawa possessing the ctx B gene of the classical biotype in Orissa, India. International Journal of Infectious Diseases. 2010; 14(5):e384-e9.

      [13] Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Taylor R, Levine MM. Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. The Journal of experimental medicine. 1988;168(4):1487-.29

      [14] Strom M, Lory S. Structure-function and biogenesis of the type IV pili. Annual Reviews in Microbiology. 1993;47(1):565-.69

      [15] LaPointe CF, Taylor RK. The type 4 prepilin peptidases comprise a novel family of aspartic acid proteases. Journal of Biological Chemistry. 2000; 275(2):1502-10.

      [16] Chaparro AP, Ali SK, Klose KE. The ToxTdependent methyl-accepting chemoreceptors AcfB and TcpI contribute to Vibrio cholerae intestinal colonization. FEMS microbiology letters. 2010; 302(2):99-105.

      [17] Sarkar A, Nandy RK, Nair GB, Ghose AC. Vibrio pathogenicity island and cholera toxin genetic element-associated virulence genes and their expression in non-O1 non-O139 strains of Vibrio cholerae. Infection and immunity. 2002; 70(8):4735-42.

      [18] Sambrook J, Russell DW. Molecular cloning: a laboratory manual: CSHL press; 2001.

      [19] Martin PR, Watson AA, McCaul TF, Mattick JS. Characterization of a five-cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa. Molecular microbiology. 1995; 16(3):497-508.

      [20] Taylor RK, Kirn TJ, Meeks MD, Wade TK, Wade WF. A Vibrio cholerae classical TcpA amino acid sequence induces protective antibody that binds an area hypothesized to be important for toxin-coregulated pilus structure. Infection and immunity. 2004;72(10):6050-60.

      [21] Hopp TP, Woods KR. Prediction of protein antigenic determinants from amino acid sequences. Proceedings of the National Academy of Sciences. 1981;78(6):3824-8.

      [22] Jertborn M, Svennerholm AM, Holmgren J. Intestinal and systemic immune responses in humans after oral immunization with a bivalent B subunit-O1/O139 whole cell cholera vaccine. Vaccine. 1996;14(15):1459-.56

      [23] Svennerholm AM, Gothefors L, Sack DA, Bardhan P, Holmgren J. Local and systemic antibody responses and immunological memory in humans after immunization with cholera B subunit by different routes. Bulletin of the World Health Organization. 1984;62(6):909-.81

      [24] Svennerholm AM, Jertborn M, Gothefors I, Karim A, Sack DA, Holmgren J. Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a cholerae. Nature Reviews Microbiology. 2005;3(8):611-20.

      [25] Qadri F, Khan AI, Faruque A, Begum YA, Chowdhury F, Nair GB, et al. Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004. Emerg Infect Dis. 2005; 11(7):1104-7.

      [26] Walker RI, Van De Verg LL, Hall RH, Schmitt CK, Woo K, Hale V. Enteric vaccines for pediatric use: Workshop summary. Vaccine. 2005; 23(46):5432-9.

      [27] Jertborn M, Svennerholm A, Holmgren J. Saliva, breast milk, and serum antibody responses as indirect measures of intestinal immunity after oral cholera vaccination or natural disease. Journal of clinical microbiology. 1986;24(2):203-9.

      [28] Lycke N, Holmgren J. Intestinal mucosal memory and presence of memory cells in lamina propria and Peyer's patches in mice 2 years after oral immunization with cholera toxin. Scandinavian journal of immunology. 1986; 23(5):611-6. combined B subunit-whole cell vaccine. Journal of Infectious Diseases. 1984; 149(6):884-93.


 

View

Download

Article ID: 20375
 
DOI: 10.14419/ijet.v7i4.7.20375




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