Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

https://doi.org/10.22146/ijbiotech.7555

Wayan T. Artama(1*), Yulia Sari(2), Didik Tulus Subekti(3), Soenarwan Hery Poerwanto(4), Jarot Subandono(5)

(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 Biology, Gadjah Mada University, Yogyakarta 55281, Indonesia
(3) Research Institute for Veterinary Science, Bogor, Indonesia
(4) Faculty of Biology, Gadjah Mada University, Yogyakarta 55281, Indonesia
(5) Faculty of Medicine, University of Surakarta, Indonesia
(*) Corresponding Author

Abstract


Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during active penetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targeted cell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasite successfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorus vacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently, this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone and sequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique. Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/  c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA was used as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor from Riboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinant plasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agar containing X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in the LB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolated using alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid was cut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward and M13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretory and secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the cloned gene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.

Keywords


Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2

Full Text:

PDF


References

Ajioka, J.W., Fitzpatrick J.M., and Reitter, C.P., 2001. Toxoplasma gondii genomics: Shedding light on pathogenesis and chemotherapy. http://www-ermm.cbcu.cam.ac.uk

Alexander, J., Jebbari, H., Bluethmann, H., Satoskar, A., and Roberts, C.W., 1996.

Immunological control of Toxoplasma gondii and appropriate vaccine design. In U. Gross, ed., Current topics in microbiology and immunology. Berlin, Heidelber: Springer-Verlag. pp.183- 195.

Ashburn, D., 1992. History and general epidemiology. In Ho-Yen, D.O. and Joss, A.W.L., eds. Human Toxoplasmosis. New York: Oxford University Press. 1-22.

Cesbron-Delauw, M.F., 1996. Role of secretory dense granule organelles in the pathogenesis of toxoplasmosis, In U. Gross, ed., Toxoplasma gondii. Berlin, Heidelberg: Springer-Verlag, pp.59-64.

Dubey, J.P., Lindsay, D.S., and Speer , C.A., 1998. Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts. Clin. Microbiol. Rev., 267-299

Dutta, C., Grimwood, J., and Kasper, L.H., 2000. Attachment of Toxoplasma gondii to a specific membrane fraction of CHO cell. Infect. Immun., 7198 – 7201.

Fuentes, I., Rubio, J.M., Ramirez, C., and Alvar, J., 2001. Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain direct analysis from clinical samples. J. Clin. Microbiol., 1566-1570

Gazzinelli, R.T., Amichay, D., Sharton- Kersten, T., Grunwald, E., Farber, J.M., and Sher. A., 1996. Role of macrophage-derived cytokines in the induction and regulation of cell- mediated immunity to Toxoplasma gondii. In U. Gross, ed., Current topics in microbiology and immunology. Berlin, Heidelberg: Springer-Verlag. pp.127-136.

Guanjin, C., Hong, G., Fangli, L.U., and Huaqin, Z., 2001. Contruction of a recombinant plasmid harbouring the rhoptry protein gene of Toxoplasma gondii and preliminary observations on DNA immunity. J. Chin. Med., 114 (8), 837 – 840.

Pfrepper, K.I., Enders, G., Gohl, M., Krczal, D., Hlobil, H., Wassenberg,D., and Soutschek, E., 2005. Seroreactivity to and avidity for recombinant antigens in toxoplasmosis. Clin. Diagn. Lab. Immunol., 12 (8), 977–982.

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

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

Yamamoto, J.H., Vallochi, A.L., Silveria, C., Filho, J.K. Nusenblatt, R.B., Neto, E.C., Gazzinelli, R.T., Belfort, R., and Rizzo, L.V., 2000. Discrimination between patients with acquired toxoplasmosis and congenital toxoplasmosis on the basis of the immune response to parasite antigens. J. Infect. Dis. 181, 2018 – 2022.



DOI: https://doi.org/10.22146/ijbiotech.7555

Article Metrics

Abstract views : 553 | views : 689

Refbacks

  • There are currently no refbacks.


Copyright (c) 2015 Indonesian Journal of Biotechnology