[Home ] [Archive]   [ فارسی ]  
:: Main :: About :: Current Issue :: Archive :: Search :: Submit :: Contact ::
:: Volume 20, Issue 1 (Bimonthly 2016) ::
Feyz 2016, 20(1): 73-80 Back to browse issues page
Expression of the protein gp40/15 Cryptosporidium parvum in E. coli
Hosein Sobati *, Habaib Jasor -Gharebagh, Hosein Honari
Health Research Center, Baqiyatallah University of Medical Sciences , sobatih@gmail.com
Abstract:   (2216 Views)

Background: Cryptosporidium is a parasitic protozoa of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts. Some of the parasite surface antigens such as gp40/15 play an important role in attaching and invasion to host cell and stimulating the immune system.  The possible access to recombinant proteins of the parasite can be provided by cloning and expression of these antigens. The aim of this study was to study the gene expression of gp40/15 Cryptosporidium parvum in Escherichia coli (E. coli).

Materials and Methods: The sequence of gene gp40/15 for Cryptosporidium parvum was extracted from GenBank (No.AF155624) and was synthetically cloned in PET28a+. The recombinant plasmid was confirmed by the colony PCR and the BamHI and XhoI restriction enzymes. The recombinant plasmid was transferred into E. coli and the protein   expression was verified using SDS-PAGE, Western blot and ELISA which was then purified by column chromatography.

Results: The results showed that the gene gp40/15 was cloned into PET28a+ plasmid. The confirmation of isolated gene cloned in PET28a+ was done by colony PCR, restriction enzymes which showed a 921bp band. The PET28a-gp40/15 plasmid expressed in E. coli showed a 43 kDa band which was confirmed using the SDS-PAGE, Western blotting and ELISA.

Conclusion: The results showed that the gene gp40/15 successfully cloned into the expression plasmid PET28a+ expressed in E.coli and recombinant protein gp40/15 is produced in it. Therefore, the recombinant protein can be used to design recombinant   vaccines and diagnostic kits in further.

Keywords: Cryptosporidium parvum, Gene gp40/15, Protein expression, Escherichia coli
Full-Text [PDF 262 kb]   (700 Downloads)    
Type of Study: Research | Subject: medicine, paraclinic
Received: 2016/04/13 | Accepted: 2016/04/13 | Published: 2016/04/13
References
1. Wang C, Luo J, Amer S, Guo Y, Hu Y, Lu Y, et al. Multivalent DNA vaccine induces protective immune responses and enhanced resistance against Cryptosporidium parvum infection. Vaccine 2011; 29(2): 323–8.
2. Petry F, Jakobi V, Tessema TS. Host immune response to Cryptosporidium parvum infection. Exp Parasitol 2010; 126(3): 304-9.
3. Singh I, Theodos C, Tzipori S. Recombinant proteins of Cryptosporidium parvum induce proliferation of mesenteric lymph node cells in infected mice. Infect Immun 2005; 73(8): 5245-8.
4. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium Pathogenicity and Virulence. Clin Microbiol Rev 2013; 26(1): 115-34.
5. Boulter-Bitzer JI1, Lee H, Trevors JT. Molecular targets for detection and immunotherapy in Cryptosporidium parvum. Biotechnol Adv 2007; 25(1): 13-44.
6. Ehigiator HN, Romagnoli P, Priest JW, Secor WE, Mead JR. Induction of murine immune responses by DNA encoding a 23-kDa antigen of Cryptosporidium parvum. Parasitol Res 2007; 101(4): 943-50.
7. Shayan P, Ebrahimzadeh E, Mokhber-Dezfouli MR, Rahbari S. Recombinant Cryptosporidium parvum p23 as a target for the detection of Cryptosporidium-specific antibody in calf sera. Parasitol Res 2008; 103(5): 1207-11.
8. Ebrahimzadeh M, Shayan P, Mokhber Dezfouli MR, Rahbari S. Recombinant Cryptosporidium parvum p23 as a Candidate Vaccine for Cryptosporidiosis. Iran J Parasitol 2009; 4(1): 1-7.
9. Borad A, Ward H. Human immune responses in cryptosporidiosis. Future Microbiol 2010; 5(3): 507-19.
10. Cevallos AM, Zhang X, Waldor MK, Jaison S, Zhou X, Tzipori S, et al. Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15. Infect Immun 2000; 68(7): 4108–16.
11. O’Connor R, Wanyiri J, Cevallos A, Priest J, Ward HD. Cryptosporidium parvum glycoprotein gp40 localizes to the sporozoite surface by association with gp15. Mol Biochem Parasitol 2007; 156(1): 80–3.
12. Wanyiri JW, O’Connor R, Allison G, Kim K, Kane A, Qiu J, et al. Proteolytic Processing of the Cryptosporidium Glycoprotein gp40/15 by Human Furin and by a Parasite-Derived Furin-Like Protease Activit. Infect Immun 2007; 75(1): 184–92.
13. O’Connor RM, Kim K, Khan F, Ward HD. Expression of Cpgp40/15 in Toxoplasma gondii: a Surrogate System for the Study of Cryptosporidium Glycoprotein Antigens. Infect Immun 2003; 71(10): 6027–34.
14. O’Connor RM, Wanyiri J, Wojczyk B, Kim K, Ward H. Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii. Mol Biochem Parasitol 2007; 152(2): 149–58.
15. Tilley M, Upton SJ, Fayer R, Barta JR, Chrisp CE, Freed PS, et al. Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum. Infect Immun 1991; 59(3): 1002–7.
16. Leav BA, Mackay MR, Anyanwu A, O’ Connor RM, Cevallos AM, Kindra G, et al. Analysis of Sequence Diversity at the Highly Polymorphic Cpgp40/15 Locus among Cryptosporidium Isolates from Human Immuno deficiency Virus-Infected Children in South Africa. Infect Immun 2002; 70(7): 3881–90.
17. Sambrook J, Russel DW. Molecular cloning: A laboratory manual, third edition. Plainview: Cold Spring Harbor Laboratory Press. Cold Spring Harbor New York; 2001.
18. Lan DT, Lan TT, Viet LQ, Quyet PV, Quang HT, Loc NH, et al. Cloning and expression of gene encoding P23 protein from Cryptosporidium parvum. J BioSci Biotech 2014; 3(3): 189-93.
19. Ahmadi AH, Honari H, Minaei ME. Cloning, Fusion, and Expression of Domain a-1 Protective Antigen (PA20) of Bacillus anthracis and N-Terminal ipaD Gene of Shigella in E. coli. Qom Univ Med Sci J 2015; 9(4): 20-9. [in Persian]
20. Bradford MM. Arapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1979; 72: 248-54.
21. Rossle NF, Latif B. Cryptosporidiosis as threatening health problem: A review. Asian Pac J Trop Biomed 2013; 3(11): 916–24.
22. Manque PA, Tenjo F, Woehlbier U, Lara AM, Serrano MG, Xu P, et al. Identification and immunological characterization of three potential vaccinogens against Cryptosporidium species. Clin Vaccine Immunol 2011; 18(11): 1796-802.
23. Allison GM, Rogers KA, Borad A, Ahmed S, Karim MM, Kane AV, et al. Antibody responses to the immunodominant Cryptosporidium gp15 antigen and gp15 polymorphisms in a case-control study of cryptosporidiosis in children in Bangladesh. Am J Trop Med Hyg 2011; 85(1): 97-104.
24. Liu K, Zai D, Zhang D, Wei Q, Han G, Gao H, et al. Divalent Cp15-23 vaccine enhances immune responses and protection against Cryptosporidium parvum infection. Parasite Immunol 2010; 32(5): 335-44.
25. Strong W B, Gut J, Nelson Richar G. Cloning and Sequence Analysis of a Highly Polymorphic Cryptosporidium parvum Gene Encoding a 60-Kilodalton Glycoprotein and Characterization of Its 15- and 45-Kilodalton Zoite Surface Antigen Products. Infect Immun 2000; 68(7): 4117–34.
Send email to the article author

Add your comments about this article
Your username or Email:

CAPTCHA


XML   Persian Abstract   Print


Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Sobati H, Jasor -Gharebagh H, Honari H. Expression of the protein gp40/15 Cryptosporidium parvum in E. coli. Feyz. 2016; 20 (1) :73-80
URL: http://feyz.kaums.ac.ir/article-1-2977-en.html


Volume 20, Issue 1 (Bimonthly 2016) Back to browse issues page
مجله علمی پژوهشی فیض ::: دانشگاه علوم پزشکی کاشان KAUMS Journal ( FEYZ )
Persian site map - English site map - Created in 0.05 seconds with 32 queries by YEKTAWEB 3921