Protein Rhopry 1 (ROP1) takizoit Toxoplasma gondii merupakan salah satu protein yang berperan penting pada proses invasi parasit ini ke dalam sel hospes. Protein tersebut diperkirakan sebagai molekul penetration-enhanching factor (PEF). Kloning gen penyandi ROP1 takizoit Toxoplasma gondii isolat RH untuk vaksinasi DNA pada mencit telah diteliti. Imunisasi DNA tersebut dapat meningkatkan respon imun seluler dan humoral. Kloning gen penyandi ROP1 takizoit Toxoplasma gondii isolat lokal belum pernah dilakukan. Tujuan penelitian ini ialah untuk memperoleh klon yang membawa gen penyandi ROP1 takizoit isolat lokal melalui teknik DNA rekombinan. Takizoit Toxoplasma gondii dikultivasi secara in vivo pada mencit strain Balb/C. Isolasi DNA dilakukan setelah beberapa kali pasase supaya diperoleh jumlah takizoit yang cukup (minimal 1x108 takizoit/ml) dan selanjutnya DNA diamplifikasi menggunakan PuRe Taq RTG-PCR Beads (Amersham Bioscience) dengan primer spesifik (Cybergene AB) dan hasilnya diligasi pada pGEM-T® Easy (Promega). Hasil ligasi kemudian ditransformasi ke dalam Escherichia coli XL-1 Blue dengan teknik heat shock dan transforman ditanam pada plate agar LB yang mengandung ampisilin, X-gal, dan IPTG. Koloni putih yang menunjukkan koloni rekombinan ditumbuhkan dalam media LB yang telah ditambahkan ampisilin pada suhu 37oC selama semalam. Plasmid DNA rekombinan diisolasi dengan metode lisis alkali dan dielektroforesis pada gel agarose 1% (SeaKem). Analisis plasmid rekombinan dilakukan dengan cara digesti menggunakan endonuklease restriksi EcoRI dan selanjutnya disekuensing untuk mengetahui urutan basanya (ABI 3130 Genetic Analyzer). Hasil penelitian menunjukkan bahwa kloning gen penyandi ROP1 Toxoplasma gondii isolat lokal menghasilkan klon yang membawa DNA insert dengan ukuran 1441 bp. DNA insert tersebut mempunyai homologi sebesar 99% dengan gen penyandi ROP1 Toxoplasma gondii isolat RH.

Rhoptry 1 protein (ROP1) of tachyzoite Toxoplasma gondii is one of proteins which play an important role during cell host invasion. It was suggested that ROP1 is a molecule related to penetrating enhancing factor (PEF). Cloning gene encoding ROP1 of tachyzoite Toxoplasma gondii RH isolate for DNA vaccination in mice has been studied. The humoral and cellular immune responses were elicited by the DNA immunization. Cloning gene encoding ROP1 of tachyzoite Toxoplasma gondii local isolate has not been studied. The aim of this study is to clone the gene encoding ROP1 protein of tachyzoite Toxoplasma gondii local isolate that has been generated by amplification using polymerase chain reaction with specific primers. DNA isolation was done after in vivo cultivation of tachyzoites in Balb/C mice and then amplified using PuRe Taq RTG-PCR Beads (Amersham Bioscience), specific primers (Cybergene AB), with positive control (RH isolate) and negative control (without template). The amplified DNA fragment was cloned into pGEM-T® Easy (Promega) and transformed into E. coli XL-1 Blue by heat shock method, then plated on LB agar containing ampicillin, X-gal and IPTG. White colonies (recombinant colonies) were picked up and put in LB medium and incubated at 37oC for overnight. Recombinant plasmids were isolated using alkali lysis method and run on a 1% agarose gel (SeaKem). Analysis of recombinant plasmids were done by digestion using restriction endonuclease EcoRI and then the recombinant plasmids were sequenced to find out the nucleotide sequence of DNA insert (ABI 3130 Genetic Analyzer). The computer software was used to analyze the DNA sequence for alignment. The result showed that cloning of the gene encoding ROP1 of tachyzoite Toxoplasma gondii local isolate produced a clone that contain DNA insert with molecular length of 1.441 kb. Sequence of those DNA insert showed 99% homology to the gene encoding ROP1 of Toxoplasma gondii RH isolate.

Kata Kunci : Kloning Gen Penyandi ROP1,Toxoplasma gondii,Vaksinasi DNA, ROP1, Toxoplasma gondii, tachyzoite, recombinant DNA