Cloning Gene Encoding Micronema 3 (Mic3) Protein of Tachyzoite Toxoplasma Gondii Local Isolate

Wayan T. Artama; Ni Nyoman Ayu Dewi; Didik Tulus Subekti

doi:10.22146/ijbiotech.7410

Cloning Gene Encoding Micronema 3 (Mic3) Protein of Tachyzoite Toxoplasma Gondii Local Isolate

https://doi.org/10.22146/ijbiotech.7410

Wayan T. Artama^(1*), Ni Nyoman Ayu Dewi⁽²⁾, Didik Tulus Subekti⁽³⁾

(1) Research Center for Biotechnology, Gadjah Mada University, Yogyakarta 55281, Indonesia; Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta 55281, Indonesia
(2) Research Center for Biotechnology, Gadjah Mada University, Yogyakarta 55281, Indonesia; Faculty of Medicine Udayana University, Denpasar 80114, Indonesia
(3) BALITVET, Bogor, Indonesia
(*) Corresponding Author

Abstract

Microneme 3 (MIC3) protein tachyzoites Toxoplasma gondii is one of protein which plays an important role during cell host invasion. Gene encoding MIC3 protein has been studied and it was suggested a potent vaccine candidate against Toxoplasma gondii infection. The aim of this research is to clone and sequence the gene encoding MIC3 protein of tachyzoites Toxoplasma gondii local isolate by amplification using polymerase chain reaction with specific primers. The amplified DNA fragment was cloned into pGEM-T and transformed into E. coli XL-1 Blue by heat shock method. Recombinant plasmids were isolated using alkali lysis method and analyzed by digestion using restriction endonuclease enzymes PstI, HindIII, NcoI and EcoRV. The recombinant plasmids then sequenced to find out the nucleotide sequence of insert gene by ABIPRISM 377 DNA Sequencer. The DNA sequence then were analyzed by computer software for alignment. The result showed that transformation in E. coli XL-1 Blue by pGEM-T produced one clone that was encoding MIC3 protein. Analysis of 489 bp from 5’ and 447 from 3’ of gene sequence showed 97-98% homology with gene encoding for MIC3 protein of RH isolate.

Keywords

MIC3 protein, Toxoplasma gondii, tachyzoite, recombinant DNA

Full Text:

PDF

References

Berg, J. M., Tymoczko, J. L. and Stryer, L., 2002. Biochemistry. 5th ed. New York: W. H. Freeman and Company, 41-73, 134- 135.

Black, M.W. and Boothroyd, J.C., 2000. Lytic cycle of Toxoplasma gondii. Microbiol. Mol. Biol. Rev., 64 (3), 607-623.

Cerede, O., Dubremetz, J. F., Bout, D. and Lebrun, M., 2002. The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesins. EMBO J., 21 (11), 2526-2536.

Cerede, O., Dubremetz, J.F., Soete, M., Deslee, D., Vial, H., Bout, D. and Lebrun, M., 2005. Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J. Exp. Med., 201 (3), 453-463.

Coppens, I. and Joiner, K.A., 2001. Parasite- host cell interactions in toxoplasmosis: new avenues for intervention? Expert review in molecular medicine. [e-book]. Cambridge University Press. Available from: http://www- ermm.cbcu.cam.ac.uk

Denkers, E.Y. and Gazzinelli, R.T., 1998. Regulation and function of T-cell- mediated immunity during Toxoplasma gondii infection. Clin. Microbiol. Rev., 11 (4), 569-588.

Gandahusada, S., 1998. Toxoplasma gondii. In Gandahusada, S., Ilahude, H.H.D., and Pribadi, W., eds. Parasitologi Kedokteran. Edisi ketiga. Jakarta: Balai Penerbit FK UI, 153-161.

Garcia-Reguet, N., Lebrun, M., Fourmaux, M.N., Mercereau-Puijalon, O., Mann, T., Beckers, C.J.M., Samyn, B., Beeumen, J.V., Bout, D. and Dubremetz, J.F., 2000. The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite. Cell. Microbiol., 2 (4), 353-364.

Ismael, A.B., Sekkai, D., Colli, C., Bout, D. and Mevelec, M.N., 2003. The MIC3 gene of Toxoplasma gondii is a novel potent vaccine candidate against toxoplasmosis. Infect. Immun., 71 (11), 6222-6228.

Prigione, I., Facchetti, P., Lecordier, L., Deslee, D., Chiesa, S., Cesbron-Delauw, M.F. and Pistoia, V., 2000. T- cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live takizoits: characterization of the fine antigenic specificity of the clones and implications for vaccine development. J. Immunol., 164, 3741-3748.

Scorza, T., Souza, D., Laloup, M., Dewit, J., Braekeleer, J.D., Verschueren, H., Vercammen, M., Huygen, K. and Jongert, E., 2003. A GRA1 DNA vaccine primes cytolytic CD8+ T cells to control acute Toxoplasma gondii infection. Infect. Immun., 71 (1), 309-316.

Sibley, L.D. and Howe, D.K., 1996. Genetic basis of pathogenecity in toxoplasmosis. In Gross, U., ed. Current Topics in Microbiology and Immunology., Berlin, Heidelberg, Springer-Verlag. 3- 13.

Vercammen, M., Scorza, T., Huygen, K., Braekeleer, J.D., Diet, R., Jacobs, D., Saman, E. and Verschueren, H., 2000. DNA vaccination with gene encoding Toxoplasma gondii antigens GRA1, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect. Immun., 68 (1), 38-45.

DOI: https://doi.org/10.22146/ijbiotech.7410