Expression of Plasmid Encoded GRA4 Gene of Toxoplasma gondii RH Strain in CHO Eukaryotic Cells
Background: Toxoplasmosis is a common infection all around the world. During pregnancy; it may lead to congenital disorders or abortion in human and animals. Severe damage of toxoplasmosis indicates to require effective vaccine. One of dense granules antigen is GRA4 that secrete from tachyzoite and bradyzoite. GRA4 genome is unique without intron and is one of the major immunogenic proteins from Toxoplasma gondii.
Methods: We confirmed the cloning of GRA4 gene into pcDNA3 by restriction enzyme and PCR of GRA4 gene with pcGRA4 plasmids as template. Then with using calcium- phosphate method we transfected the pcGRA4 into CHO (Chinesehamster ovary) cells. The yielded protein was separated by SDS-PAGE and moved by electroblotting to nitrocellulose paper.
Results: Result of SDS-PAGE analysis showed the appearance of band approximately 42 kDa which was absent in the negative control, that was able to identify toxoplasmosis antibody IgM+ serum in western blot analysis.
Conclusion: pcGRA4 plasmid is able to synthesis of antigenic protein in CHO cells. The ability of pcGRA4 for induction of protective immune response against toxoplasmosis will be evaluated in mouse model.
2. Kravetz JD, Federman DG. Prevention of toxo-plasmosis in pregnancy: knowledge of risk factors. Infect Dis Obstet Gynecol. 2005; 13(3): 161-5.
3. Kijlstra A, Jongert E. Control of the risk of human toxoplasmosis transmitted by meat. Int J Parasitol. 2008;38(12): 1359-70.
4. Meerburg BG, Van Riel JW, Cornelissen JB et al. Cats and goat whey associated with Toxoplasma gondii infection in pigs. Vector Borne Zoonotic Dis. 2006;6(3): 266-74.
5. Suzuki Y, Orellana MA, Schreiber RD, Remington JS. Interferon-gamma: the major mediator of re-sistance against Toxoplasma gondii. Science. 1988;240(4851): 516-8.
6. Buxton D. Toxoplasmosis. In: Diseases of Sheep. 2nd edition. Ed. W. B. Martin and I. D. Aitken. pp. 49-58. Blackwell Scientific Publications, Oxford. (1991).
7. Mévélec MN, Ducournau C, Bassuny Ismael A et al. Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep. Vet Res. 2010;41(4): 49.
8. Son ES, Nam HW. Detection and characterization of excretory/secretory proteins from Toxoplasma gondii by monoclonal antibodies. Korean J Parasitol. 2001; 39(1): 49–56.
9. Mercier C, Adjogble KD, Daubener W, Delauw MF. Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites? Int J Parasitol. 2005; 35(8): 829-849.
10. Cesbron-Delauw MF,Gendrin C, Travier L et al. Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole. Traffic. 2008; 9: 657–664.
11. Cornelissen AWCA, Overdulve JP, Ploeg MVD. Determination of nuclear DNA of five eucoccidian parasites, Isospora (Toxoplasma) gondii, Sarcocystis cruzi, Eimeria tenella, E. acervulina and Plasmodium berghei, with special reference to gamontogenesis and meiosis in I. (T.) gondii. Parasitology. 1984; 88: 531-553.
12. Chardes T, Bourguin I, Mevelec MN et al. Anti-body responses to Toxoplasma gondii in sera, intestinal secretions, and milk from orally infected mice and characterization of target antigens. Infect Immun. 1990; 58(5): 1240–1246.
13. Meisel R, Stachelhaus S, Mevelec MN et al. Identifi-cation of two alleles in the GRA4 locus of Toxo-plasma gondii determining a differential epitope which allows discrimination of type I versus type II and III strains. Mol Biochem Parasitol. 1996; 81(2): 259–263.
14. Mevelec M, Bout D, Desolme B et al. Evaluation of protective effect of DNA vaccination with genes encoding antigen GRA4and SAG1associated with GM-CSF plasmid antigen acute, chronical and congenital Toxoplasmosis in mice. Vaccine. 2005;23: 4489-4499.
15. Martin V, Supanitsky A, Echeverria PC et al. Re-combinant GRA4 or ROP2 protein combined with alum or the GRA4 gene provides partial pro-tection in chronic murine models of Toxoplasmo-sis. Clin and Diagnlab Immunol. 2004; 11(4): 704-710.
16. Zhang G, Huong VT, Battur B. A hetrologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, induced protective immunity against Toxoplasma gondii infec-tion in mice. Parasitology. 2007; 134(10): 1339-1346.
17. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: A laboratory manual, Second Edition. Plainview: Cold Spring Harbor Laboratory Press. 1989.
18. Masebi N, Ghafarifar F, Sharifi Z et al. Cloning and Characterization of GRA4 gene of Toxoplasma gondii (RH strain) in expression eukaryotic vector pcDNA3. Daneshvar. 2010;17(85): 1-8.
19. Martin J, Annette SchallhornM, F M Wurm. Transfecting mammalian cells: optimization of criti-cal parameters affecting calcium-phosphate precipi-tate formation. Nucleic Acids Res. 1996; 24(4): 596-601.
20. Liu Q, Singla LD, Zhou H. Vaccines against Tox-oplasma gondii: status, challenges and future direc-tions. Hum Vaccin Immunother. 2012; 8(9): 1305–1308.
21. Jongert E, Roberts C W, Gargano N et al. Vac-cines against Toxoplasma gondii: challenges and op-portunities. Mem Inst Oswaldo Cruz. 2009;104(2): 252-266,
22. Chardes T, Velge-Roussel F, Mevelec P et al. Mu-cosal and systemic cellular immune responses in-duced by Toxoplasma gondii antigens in cyst orally in-fected mice. Immunology. 1993; 78: 421-429.
23. Ho-Woo Nam. GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane. Korean J Parasitol. 2009; 47: S29-S37.
24. RamH, Rao JR,Tewari AK et al. Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii. Parasitol Res. 2013; 112: 2487–2494.
25. Min Meng, AihuaZ, GangLu et al. DNA prime and peptide boost immunizationprotocol encod-ing the Toxoplasma gondii GRA4 induces strong pro-tective immunity in BALB/c mice. BMC Infect Dis. 2013; 13: 494.
26. Lau YL,Hasan MT, Thiruvengadam G et al. Clon-ing and expression of Toxoplasma gondii dense granular protein 4 )GRA4( in Pichiapastoris. Trop Biomed. 2010;27)3(: 525–533.
27. Delsolme B, Mevelec MN, Buzoni-Gatel D, Bout D. Induction of protective immunity against Tox-oplasmosis in mice by DNA immunization with a plasmid encoding Toxoplasma gondii GRA4 gene. Vaccine. 2000; 18: 2512-2521.
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|Toxoplasma gondii GRA4 gene Gene expression CHO cell|
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