Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

Document Type: Original Article

Authors

1 ¹ Department of Biotechnology and Microbiology, School of Medicine, Arak University of Medical Sciences, Arak, Iran

2 Department of Microbiology and Immunology, School of Medicine, Arak University of Medical Sciences, Arak, Iran

3 ³Biology Department, Payame Noor University, Arak, Iran

4 Department of Microbiology, Islamic Azad University, Qom Branch, Qom, Iran

5 Molecular and Medicine Research Center, Department of Microbiology, School of Medicine, Arak University of Medical Sciences, Arak, Iran

Abstract

Objective(s):
Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. 
 
Materials and Methods:
The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification.
Results:
Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera.
Conclusion
: Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

Keywords


1. Cover TL, Blaser MJ. Helicobacter pylori and gastroduodenal disease. Annu Rev Med 1992; 43: 135-145.

2. Atherton JC, Blaser MJ. Coadaptation of Helicobacter pylori and humans: ancient history, modern implications. J Clin Invest 2009; 119: 2475-2487.

3. Forman D. Helicobacter pylori and gastric cancer. Scand J Gastroenterol 1996; 31:23-26.

4. Sitas F, Forman D, Yarnell JWG, Burr ML, Elwood PC, Pedley S, et al. Helicobacter pylori infection rates in relation to age and social class in a population of Welsh men. Gut 1991; 32:25-28.

5. MullerI, Medina-selby A, Placios JL, Martinez P, Opazo P, Bruce E, et al. Cloning and comparison of ten gene sequences of a Chilean Helicobacter pylori strain with other Helicobacter pylori strains revealed higher variability for VacA and CagA virulence factors. Biol Res 2002; 35:67-84.

6. Cover TL, Blanke SR. Helicobacter pylori VacA, a paradigm for toxin multifunctionality. Nat Rev Microbiol 2005; 3:320-332.

7. de Bernard M, Cappon A, Del Giudice G, Rappuoli R, Montecucco C. The multiple cellular activities of the VacA cytotoxin of Helicobacter pylori. Int J Med Microbiol 2004; 293:589-597.

8. Gangwer KA, Shaffer CL, Suerbaum S, Lacy DB, Cover TL, Bordenstein SR. Molecular Evolution of the Helicobacter pylori Vacuolating Toxin Gene VacA. J Bacteriol 2010; 192:6126-6135.

9. Isomoto H, Moss J, Hirayama T. Pleiotropic action of Helicobacter pylori vacuolating cytotoxin, VacA. Tohoku J Exp Med 2010; 220: 3-14.

10. Papine E, Zoratti M, Cover TL. In search of the Helicobacter pylori VacA mechanism of action. Toxicon 2001; 39:1757-1767.

11. Wada A, Yamasaki E, Hirayama T. Helicobacter pylori vacuolating cytotoxin, vacA, is responsible for gastric ulceration. J Biochem 2004; 136:741-746.

12. Talebkhan Y, Mahboudi F, Sarrami R, Barkhordari F, Amani M, Mohammadi M. Cloning and expression of the heterogenic vacuolating cytotoxin from an Iranian Helicobacter pylori strain. Iran J Biotechnol 2004; 2:123-131.

13. Mohammadi M, Oghalaie A, Mohajerani N, Massarrat S

, Nasiri M, Bonnedsen M, et al. Helicobacter pylori vacuolating cytotoxin and its allelic mosaicism as a predictive marker for Iranian dyspeptic patients. Bull Soc Pathol Exot 2003; 96: 3-5.

14. Kolaskar AS, Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 1990; 276:172-174.

15. Sambrook J, Russel D. Molecular cloning: A Laboratory Manual. 3

rd ed., New York: Cold Spring Harbor Laboratory Press; 2001.

16. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463-5467.

17. Wheeldon TU, Hoang TT, Phung DC, Bjorkman A, Granstrom M, Sorberg M. Long-term follow-up of Helicobacter pylori eradication therapy in Vietnam: reinfection and clinical outcome. Aliment Pharmacol Ther 2005; 21:1047-1053.

18. Garner JA, Cover TL. Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells. Infect Immun 1996; 64:4197-4203.

19. Rudi J, Kolb C, Maiwald M, Zuna I, von Herbay A, Galle PR, et al. Serum antibodies against the Helicobacter pylori proteins CagA and VacA are associated with an increased risk for gastric adenocarcinoma. Dig Dis Sci 1997; 42:1652-1659.

20. Phadnis SH, Ilver D, Janzon L, Normark S, Westblom TU. Pathological significance and molecular characterization of the vacuolating toxin gene of Helicobacter pylori. Infect Immun 1994; 62:1557-1565.

21. Nguyen VQ, Caprioli RM, Cover TL. Carboxy-terminal proteolytic processing of Helicobacter pylori vacuolating toxin. Infect Immun 2001; 69: 543-546.

Hasanzadeh et al Expression and Antigenic Evaluation of VacA

22. Sewald X, Fischer W, Haas R. Sticky socks: Helicobacter pylori VacA takes shape. Trends Microbiol 2008; 16:89-92.

23. Gangwer KA, Mushrush DJ, Stauff DL, Spiller B, McClain MS, Cover TL, et al. Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain. Proc Natl Acad Sci USA 2007; 104:16293-16298.

24. Liu KY, Shi Y, Luo P, Yu SH, Chen L, Zhao ZH, et al. Therapeutic efficacy of oral immunization with attenuated Salmonella typhimurium expressing Helicobacter pylori CagA, VacA and UreB fusion proteins in mice model. Vaccine 2011; 2:6679

-6685.

25. Ivie SE, McClain MS, Torres VJ, Scott Algood HM, Lacy DB, Yang R, et al. Helicobacter pylori VacA subdomain required for intracellular toxin activity and assembly of functional oligomeric complexes. Infect Immun 2008; 76:2843-2851.

26. Sugimura K, Higashi N. A novel outer-membrane-associated protease in Escherichia coli. J Bacteriol 1988; 170:3650-3654.

27. McCormick M, Mierendorf R. S.Tag: A multipurpose fusion peptide for recombinant proteins. inNovations 1994; 1: 4-7.

28. Abtahi H, Salmanian AH, Rafati S, Behzadian Nejad G, Hassan ZM. High level expression of recombinant ribosomal protein (L7/L12) from Brucella abortus and its reaction with infected human sera. Iran Biomed J 2004; 8:13-18.