Volume 20, Issue 3 (Iranian South Medical Journal 2017)
Iran South Med J 2017, 20(3): 245-256 |
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Sadeghi M, Doosti A. Cloning and Study of Expression of Helicobacter Pylori FlaAGene in Eukaryotic System. Iran South Med J 2017; 20 (3) :245-256
URL: http://ismj.bpums.ac.ir/article-1-876-en.html
URL: http://ismj.bpums.ac.ir/article-1-876-en.html
1- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
2- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran ,abbasdoosti@yahoo.com
2- Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran ,
Abstract: (5610 Views)
Background: Helicobacter pylori is the most common bacterium causing chronic infections worldwide. Expression of flagella and bacterial motility are essential in colonization and virulence. FlaA gene is one of the flagellin-encoding genes that play the key role in the colonization and bacterial motility, and it has a significant impact on immunization. This study aimed to design, construct and evaluate the Helicobacter pylori flaA gene expression in eukaryotic cells.
Material and Methods: In this experimental study, genomic DNA was purified from the Helicobacter pylori standard strain, and flaA gene was amplified and isolated by PCR method with using the specific primers. Then, this gene was cloned into pTZ vector by T/A cloning technique. To express flaA gene and generate the final construct, the flaA gene was removed from pTZ plasmid and subcloned into the pcDNA3.1 (-) expression vector. The pcDNA3.1 (-)-flaA construct was transformed into CHO cells by electroporation, and flaA eukaryotic gene expression was studied using the SDS-PAGE method.
Results: The results showed that flaA gene PCR product was cloned into pTZ vector and amplified in Escherichia coli TOP10F strain. Also, the enzymatic digestion and sequencing showed that the pcDNA3.1 (-)-flaA was performed. Finally, the evaluation of the Helicobacter pyloriflaA gene expression in CHO cells showed that the generated gene construct could express the flaA product in a eukaryotic system, successfully.
Conclusion: Given that the pcDNA3.1 (-)-flaA as a final construct can express the flaA protein of the Helicobacter pylori in animal cells. Flagellin protein is one of the essential antigens of the bacterium So we can appropriately say that gene pcDNA3.1(-)-flaA construct is a suitable candidate for usage in the field of gene vaccines against Helicobacter pylori and it can be used to check the immunization in the laboratory animals in the future research.
Type of Study: Original |
Subject:
Biochemistry. Cell Biology and Genetics
Received: 2016/10/3 | Accepted: 2017/01/9 | Published: 2017/07/5
Received: 2016/10/3 | Accepted: 2017/01/9 | Published: 2017/07/5
References
1. Doosti A, Rahimian GA, Nassiri J, et al. Prevalence of the cagA-positive Helicobacter pylori strains isolated from gastric biopsy specimens in Shahrekord. Armaghane Danesh 2007; 12(1): 29-38. (Persian)
2. Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006; 19(3): 449-90. [DOI:10.1128/CMR.00054-05]
3. Souod N, Kargar M, Doosti A, et al. Genetic Analysis of cagA and vacA Genes in Helicobacter Pylori Isolates and their relationship with Gastroduodenal diseases in the west of Iran.Iran Red Crescent Med J 2013; 15(5): 371-5. [DOI:10.5812/ircmj.3732]
4. Pakbaz Z, Shirazi MH, Pourmand MR, et al. Evaluation of Rapid Urease Test Compared with Polymerase Chain Reaction for Diagnosis of Helicobacter pylori. Iran South Med J 2014; 16(6): 394-400. (Persian).
5. Kargar M, Ghorbani-Dalini S, Doosti A, et al. Real-Time PCR assay using allele-specific TaqMan probe for detection of clarithromycin resistance and its point mutations in Helicobacter pylori. J Isfahan Med Sch 2011; 29(126): 65-73. (Persian) [DOI:10.5772/22655]
6. Haggerty TD, Perry S, Sanchez L, et al. Significance of transiently positive enzyme-linked immunosorbent assay results in detection of Helicobacter pylori in stool samples from children. J Clin Microbiol 2005; 43(5): 2220-3. [DOI:10.1128/JCM.43.5.2220-2223.2005]
7. Kargar M, Baghernejad M, Doosti A. Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains.Daru 2010; 18(2): 137-40.
8. Haiko J, Westerlund-Wikström B.The role of the bacterial flagellum in adhesion and virulence.Biology (Basel) 2013; 2(4): 1242-67. [DOI:10.3390/biology2041242]
9. Kikuchi S, Crabtree JE, Forman D, et al. Association between infections with CagA-positive or -negative strains of Helicobacter pylori and risk for gastric cancer in young adults. Research Group on Prevention of Gastric Carcinoma Among Young Adults. Am J Gastroenterol 1999; 94(12): 3455-9. [DOI:10.1111/j.1572-0241.1999.01607.x]
10. Kao CY, Sheu BS, Wu JJ. CsrA regulates Helicobacter pylori J99 motility and adhesion by controlling flagella formation. Helicobacter 2014; 19(6): 443-54. [DOI:10.1111/hel.12148]
11. Tomb JF, White O, Kerlavage AR, et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 1997; 388(6642): 539-47. [DOI:10.1038/41483]
12. Chaban B, Ng SY, Kanbe M, et al. Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis. Mol Microbiol 2007; 66(3): 596-609. [DOI:10.1111/j.1365-2958.2007.05913.x]
13. Häse CC. Analysis of the role of flagellar activity in virulence gene expression in Vibrio cholerae. Microbiology 2001; 147(Pt 4): 831-7. [DOI:10.1099/00221287-147-4-831]
14. Mizel SB, Bates JT. Flagellin as an adjuvant: cellular mechanisms and potential. J Immun 2010; 185(10): 5677-82. [DOI:10.4049/jimmunol.1002156]
15. Lee SK, Stack A, Katzowitsch E, et al. Helicobacter pylori flagellins have very low intrinsic activity to stimulate human gastric epithelial cells via TLR5. Microbes Infect 2003; 5(15): 1345-56. [DOI:10.1016/j.micinf.2003.09.018]
16. Wheeldon TU, Hoang TT, Phung DC, et al. Long-term follow-up of Helicobacter pylori eradication therapy in Vietnam: reinfection and clinical outcome. Aliment Pharmacol Ther 2005; 21(8): 1047-53. [DOI:10.1111/j.1365-2036.2005.02408.x]
17. Doosti A, Ghaasemi-Dehkordi P, Kargar M, et al. Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (stx2) gene. Genetika 2015; 47(2): 499-507. [DOI:10.2298/GENSR1502499D]
18. Doosti A. Cloning of the gene encoding neurotoxin heavy chain of Clostridium botulinum in E. coli. J Microbial World 2013; 5(3): 77-84. (Persian)
19. Talebi Bezmin Abadi A. Vaccine against Helicobacter pylori: inevitable approach. World J Gastroenterol 2016; 22(11): 3150-7. [DOI:10.3748/wjg.v22.i11.3150]
20. Kabir S. The current status of Helicobacter pylori vaccines: a review. Helicobacter 2007; 12(2): 89-102. [DOI:10.1111/j.1523-5378.2007.00478.x]
21. Yan J, Liang SH, Mao YF, et al. Construction of expression systems for flaA and flaB genes of Helicobacter pylori and determination of immunoreactivity and antigenicity of recombinant proteins. World J Gastroenterol 2003; 9(10): 2240-50. [DOI:10.3748/wjg.v9.i10.2240]
22. Mori J, Vranac T, Smrekar B, et al. Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori. Vaccine 2012; 30(40): 5856-63. [DOI:10.1016/j.vaccine.2012.07.011]
23. Yeh HY, Hiett KL, Line JE, et al. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins. Microbiol Res 2013; 168(4): 192-8. [DOI:10.1016/j.micres.2012.11.010]
24. Yuan Y, Wang X, Guo S, et al. Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus. Chinese J Oceanol Limnol 2010; 28(6): 1254-60. [DOI:10.1007/s00343-010-0068-1]
25. Doosti A, Rahimian GA, Nassiri J, et al. Prevalence of the cagA-positive Helicobacter pylori strains isolated from gastric biopsy specimens in Shahrekord. Armaghane Danesh 2007; 12(1): 29-38. (Persian)
26. Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006; 19(3): 449-90. [DOI:10.1128/CMR.00054-05]
27. Souod N, Kargar M, Doosti A, et al. Genetic Analysis of cagA and vacA Genes in Helicobacter Pylori Isolates and their relationship with Gastroduodenal diseases in the west of Iran.Iran Red Crescent Med J 2013; 15(5): 371-5. [DOI:10.5812/ircmj.3732]
28. Pakbaz Z, Shirazi MH, Pourmand MR, et al. Evaluation of Rapid Urease Test Compared with Polymerase Chain Reaction for Diagnosis of Helicobacter pylori. Iran South Med J 2014; 16(6): 394-400. (Persian).
29. Kargar M, Ghorbani-Dalini S, Doosti A, et al. Real-Time PCR assay using allele-specific TaqMan probe for detection of clarithromycin resistance and its point mutations in Helicobacter pylori. J Isfahan Med Sch 2011; 29(126): 65-73. (Persian) [DOI:10.5772/22655]
30. Haggerty TD, Perry S, Sanchez L, et al. Significance of transiently positive enzyme-linked immunosorbent assay results in detection of Helicobacter pylori in stool samples from children. J Clin Microbiol 2005; 43(5): 2220-3. [DOI:10.1128/JCM.43.5.2220-2223.2005]
31. Kargar M, Baghernejad M, Doosti A. Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains.Daru 2010; 18(2): 137-40.
32. Haiko J, Westerlund-Wikström B.The role of the bacterial flagellum in adhesion and virulence.Biology (Basel) 2013; 2(4): 1242-67. [DOI:10.3390/biology2041242]
33. Kikuchi S, Crabtree JE, Forman D, et al. Association between infections with CagA-positive or -negative strains of Helicobacter pylori and risk for gastric cancer in young adults. Research Group on Prevention of Gastric Carcinoma Among Young Adults. Am J Gastroenterol 1999; 94(12): 3455-9. [DOI:10.1111/j.1572-0241.1999.01607.x]
34. Kao CY, Sheu BS, Wu JJ. CsrA regulates Helicobacter pylori J99 motility and adhesion by controlling flagella formation. Helicobacter 2014; 19(6): 443-54. [DOI:10.1111/hel.12148]
35. Tomb JF, White O, Kerlavage AR, et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 1997; 388(6642): 539-47. [DOI:10.1038/41483]
36. Chaban B, Ng SY, Kanbe M, et al. Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis. Mol Microbiol 2007; 66(3): 596-609. [DOI:10.1111/j.1365-2958.2007.05913.x]
37. Häse CC. Analysis of the role of flagellar activity in virulence gene expression in Vibrio cholerae. Microbiology 2001; 147(Pt 4): 831-7. [DOI:10.1099/00221287-147-4-831]
38. Mizel SB, Bates JT. Flagellin as an adjuvant: cellular mechanisms and potential. J Immun 2010; 185(10): 5677-82. [DOI:10.4049/jimmunol.1002156]
39. Lee SK, Stack A, Katzowitsch E, et al. Helicobacter pylori flagellins have very low intrinsic activity to stimulate human gastric epithelial cells via TLR5. Microbes Infect 2003; 5(15): 1345-56. [DOI:10.1016/j.micinf.2003.09.018]
40. Wheeldon TU, Hoang TT, Phung DC, et al. Long-term follow-up of Helicobacter pylori eradication therapy in Vietnam: reinfection and clinical outcome. Aliment Pharmacol Ther 2005; 21(8): 1047-53. [DOI:10.1111/j.1365-2036.2005.02408.x]
41. Doosti A, Ghaasemi-Dehkordi P, Kargar M, et al. Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (stx2) gene. Genetika 2015; 47(2): 499-507. [DOI:10.2298/GENSR1502499D]
42. Doosti A. Cloning of the gene encoding neurotoxin heavy chain of Clostridium botulinum in E. coli. J Microbial World 2013; 5(3): 77-84. (Persian)
43. Talebi Bezmin Abadi A. Vaccine against Helicobacter pylori: inevitable approach. World J Gastroenterol 2016; 22(11): 3150-7. [DOI:10.3748/wjg.v22.i11.3150]
44. Kabir S. The current status of Helicobacter pylori vaccines: a review. Helicobacter 2007; 12(2): 89-102. [DOI:10.1111/j.1523-5378.2007.00478.x]
45. Yan J, Liang SH, Mao YF, et al. Construction of expression systems for flaA and flaB genes of Helicobacter pylori and determination of immunoreactivity and antigenicity of recombinant proteins. World J Gastroenterol 2003; 9(10): 2240-50. [DOI:10.3748/wjg.v9.i10.2240]
46. Mori J, Vranac T, Smrekar B, et al. Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori. Vaccine 2012; 30(40): 5856-63. [DOI:10.1016/j.vaccine.2012.07.011]
47. Yeh HY, Hiett KL, Line JE, et al. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins. Microbiol Res 2013; 168(4): 192-8. [DOI:10.1016/j.micres.2012.11.010]
48. Yuan Y, Wang X, Guo S, et al. Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus. Chinese J Oceanol Limnol 2010; 28(6): 1254-60. [DOI:10.1007/s00343-010-0068-1]
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