Kajian Serologis Toksoplasmosis pada Sapi dengan Metode Enzyme Linked Immunosorbent Assay Menggunakan Protein Rekombinan GRA-1.
WIHADMADYATAMI, Hevi , Wayan T. Artama
2009 | Tesis |Toksoplasmosis merupakan penyakit karena infeksi protozoa intraseluler Toxoplasma gondii. Usaha untuk mengendalikan penyakit ini telah banyak dilakukan namun belum menunjukkan hasil yang memuaskan. Antigen GRA-1 merupakan protein yang diproduksi oleh takizoit dan bradizoit Toxoplasma gondii dan dapat digunakan sebagai perangkat diagnostik dan kandidat vaksin karena memiliki kemampuan untuk menginduksi respon imun humoral dan seluler pada mencit dan manusia. Protein ini dapat diisolasi dari parasit tetapi untuk mendapatkan kuantitas yang mencukupi masih menghadapi kendala. Kloning gen GRA-1 pada vektor ekspresi pET-32a(+) telah berhasil dilakukan (Widayanti, 2008). Tujuan penelitian ini adalah untuk memproduksi protein GRA-1 melalui teknik rekombinan dengan melakukan isolasi protein rekombinan GRA-1 takizoit Toxoplasma gondii isolat lokal dan menerapkan protein tersebut dalam kajian serologis prevalensi toksoplasmosis pada sapi. Gen penyandi GRA-1 diekspresikan pada bakteri E. coli BL21 yang telah disisipi gen GRA-1 menggunakan plasmid pET-32a(+) ke dalam media LB yang mengandung ampisilin dan IPTG. Protein kemudian diisolasi, diidentifikasi dengan SDS-PAGE dan dilanjutkan dengan purifikasi protein. Hasil purifikasi protein rekombinan dielektroforesis kembali, diidentifikasi dengan imunobloting. Protein rekombinan GRA-1 dianalisis kesesuaiannya untuk menggantikan protein ESA menggunakan agreement between two test (kappa). Protein rekombinan GRA-1 kemudian digunakan dalam kajian serologis prevalensi toksoplasmosis pada sapi menggunakan metode ELISA. Sampel serum sapi diperoleh dari Sleman, Magelang, Bali, Jawa Barat, Sumatera Utara, Sumatera Barat, Sumatera Selatan, Malang, dan Sulawesi Selatan masing-masing sebanyak 57 sampel, 56 sampel, 125 sampel, 90 sampel, 50 sampel, 107 sampel, 56 sampel, 52 sampel, dan 258 sampel. Hasil penelitian menunjukkan bahwa protein GRA-1 telah berhasil diproduksi dan di purifikasi dengan berat molekul 24 kDa. Identifikasi dengan imunobloting menunjukkan adanya reaksi antigen antibodi antara antibodi poliklonal anti ESA dan antibodi poliklonal anti GRA-1 dengan protein rekombinan GRA-1. Protein rekombinan GRA-1 mempunyai kemampuan untuk menggantikan protein ESA dengan nilai agreement between two test (kappa) 0,78. Kajian serologis menunjukkan prevalensi toksoplasmosis di Sleman, Magelang, Bali, Jawa Barat, Sumatera Utara, Sumatera Barat, Sumatera Selatan, Malang, dan Sulawesi Selatan adalah 21,05%, 39,28%, 16%, 18%, 18,7%, 3%, 11%, 46,15% dan 9,3%. Kata kunci: T. gondii, protein rekombinan GRA-1, ELISA, seroprevalensi
Toxoplasmosis is a disease caused by intracellular protozoa so called Toxoplasma gondii. Many efforts have been done to overcome this disease, but unfortunately it has not showed many satisfactory results. GRA-1 is an antigen produced by takyzoite and bradyzoite of Toxoplasma gondii. This protein can be used as diagnostic and vaccine candidate, because GRA-1 has capability to induce humoral and cellular immune respon in both human and mice. This protein can be isolated directly from the parasite but it will have many problems to get enough quantity. The cloning of GRA-1 gene in expression vector of pET-32a(+) has been successfully done (Widayanti, 2008). The purpose of this research is to produce GRA-1 protein using recombinant technique by isolating protein of GRA-1 takyzoite local isolate of Toxoplasma gondii and to apply this protein for determining the seroprevalence of bovine toxoplasmosis in Indonesia. Escherichia coli bacteria BL21 that was already transformed with pRT- 32a(+) recombinant grown in LB medium containing ampicillin and IPTG, and then these bacteria exposed GRA-1 protein in a large amounts. By using a standar procedure recombinant protein was isolated from bacteria using affinity chromatography of nichel-TED. The result of purification of recombinant protein will be electroforized again and identified by using immunoblotting. The GRA-1 recombinant protein compatibility is analyzed to change ESA protein using agreement between two tests (kappa), and then recombinant protein of GRA-1 is used to identify toxoplasmosis seroprevalence in bovine by using ELISA method. The sample of Bovine serum is collected from several regions namely Sleman, Magelang, Bali, West Java, North Sumatera, West Sumatera, South Sumatera, Malang and South Sulawesi, a number of serum samples for the following region are 57 samples, 56 samples, 90 samples, 50 samples, 107 samples, 56 samples, 125 samples, 52 samples, and 258 samples. The results of this research show that gene encoding of GRA-1 protein has been successfully expressed. GRA-1 protein has been successfully isolated and purified with molecular weight 24 kDa. Identificatio n using immunoblotting shows that there is antigen antibody reaction between polyclonal antibody anti ESA or anti GRA-1 and GRA-1 recombinant protein. Recombinant protein of GRA-1 has capability to replace ESA protein with agreement between two test (kappa) value is 0,78. Serological study shows that the percentage of prevalence of bovine toxoplasmosis on Sleman, Magelang, Bali, Jawa Barat, Sumatera Utara, Sumatera Barat, Sumatera Selatan, Malang and Sulawesi Selatan are 21,05%, 39,28%, 16%, 18%, 18,7%, 3%, 11%, 46,15% and 9,3%. Keywords: T. gondii, GRA-1 recombinant protein, ELISA, seroprevalence
Kata Kunci : T. gondii, protein rekombinan GRA-1, ELISA, seroprevalensi
