Kloning dan identifikasi cDNA penyandi protein ESA (Axcretory-Secretory Antigens) Takizoit Toxoplasma gondii isolat lokal

SARI, Yulia, Dr.drh. Wayan T. Artama

2006 | Tesis | S2 Bioteknologi

Excretory and Secretory Antigens (ESA) berperan pada proses penetrasi aktif dan modifikasi vakuola parasitoporus dalam sel target. ESA menyebabkan Toxoplasma mampu melakukan penetrasi secara aktif sebelum sel target memberikan respon. Modifikasi vakuola intraseluler mengakibatkan takizoit dari Toxoplasma gondii akan mampu bertahan hidup dan berkembang biak tanpa hambatan. Tujuan penelitian ini adalah melakukan kloning dan identifikasi cDNA penyandi ESA T. gondii isolat lokal. Ribonucleic Acid (RNA) total diisolasi dari takizoit T. gondii isolat lokal yang diperbanyak secara in vivo pada mencit strain Balb/c. Messenger RNA diisolasi dari RNA total dengan menggunakan PolyATract mRNA Isolation System (Promega). Messenger RNA digunakan untuk sintesis cDNA dengan menggunakan Riboclone® cDNA Synthesis System AMV-RT (Promega). Complementary DNA yang didapat ditambahkan EcoRI adaptor menggunakan Riboclone® EcoRI Adaptor Ligation System (Promega) dan diligasi pada pUC19. Plasmid rekombinan ditransformasi ke Escherichia coli XL1-Blue. Hasil transformasi ditumbuhkan pada plat agar yang mengandung X-Gal, IPTG dan ampisilin. Koloni rekombinan (koloni putih) ditumbuhkan dalam media LB dan ampisilin pada suhu 37oC semalam. Plasmid DNA rekombinan diisolasi dengan metode lisis alkali dan dielektroforesis pada gel agarose 1%. Rekombinan yang membawa cDNA penyandi ESA takizoit T. gondii isolat lokal diidentifikasi dengan immunoblotting menggunakan poliklonal ESA. Analisis insert dilakukan dengan pemotongan menggunakan EcoRI selanjutnya disequencing menggunakan Big Dye Terminator Mix AB1 377A Sequencer dengan primer M13 Forward dan primer M13 Reverse. Hasil penelitian menunjukkan bahwa transformasi pada E. coli XL1-Blue dengan menggunakan vektor pUC19 didapat satu rekombinan menyandi ESA Toxoplasma gondii isolat lokal. Hasil imunobloting mengindentifikasikan klon rekombinan penyandi ESA T. gondii isolat lokal mengekspresikan protein dengan berat molekul 54 kDa (p54). Hasil sekuensing dari klon rekombinan yang membawa gen penyandi ESA merupakan gen penyandi protein roptri 2.

Excretory and secretory antigen (ESA) play an important role during active penetration process and parasitophorus vacuole modification at cell target. Excretory and secretory antigen protein induces Toxoplasma gondii to be able to actively penetrate targeted cell, in addition, ESA protein stimulates intracellular vacuole modification. It is, therefore, parasitophorus vacuole is unable to fuse with other intracellular compartments such as lisosomal vacuole. Consequently, Toxoplasma gondii will be able to survive and multiply. The current study is aimed to klon and to identify cDNA encoding ESA of local isolated T. gondii takizoit through DNA recombinant technique. Total ribonucleic acid (RNA) was isolated from tachizoites of local isolated T. gondii that were grown up in mice (Balb/c). Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System (Promega). Messenger RNA were used to synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT (Promega). Complementary DNA through EcoRI adaptor of Riboclone EcoRI Adaptor Ligation System (Promega) 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 put in the LB medium and incubated 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order to identify cDNA recombinant with that express ESA of T. gondii local isolate. Plasmid recombinant were isolated using alkali lysis methode and were elektroforated in 1% agarose gel. DNA recombinant plasmid was cut using Eco RI and sequencing through Big Dye Terminator Mix AB1 377A Sequencer with M13 Forward and M13 Reverse. 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 alligment of sequence from the cloned gene indicated that 98% homology with gen encoding for ROP-2 of Toxoplasma gondii RH isolate.

Kata Kunci : Kloning,Identifikasi cDNA,ESA,Toxoplasma Gondii,ESA, Toxoplasma gondii, takizoit, complementary DNA