Abstract
Microneme protein (MIC) is one of proteins that belongs to excretory-secretory antigens (ESAs) of Toxoplasma gondii. Microneme 3 protein (MIC-3) is the protein that plays an important role in the invasion proccess during cell infection as a mediator attachment parasite to the host cell. The aim of this research is to clone mic3 (gene encoding for MIC-3) of T. gondii from local isolate using recombinant DNA technology by cloning mic3 in an expression vector. Deoxyribonucleic acid (DNA) from T. gondii tachyzoites was amplified by PuRe Taq RTG-PCR Beads using mic3 specific primers. Amplified DNA was double digested using EcoRV and HindIII restriction endonucleases and then purified using EZ-10 spin coloumn purification kit. The mic3 DNA was ligated into pET-32a(+) expression vector and transformated into Escherichia coli BL21. The results showed that recombinant mic3gene 4.2 kDa has been successfully performed by cloning gene encoding for MIC-3 protein of T. gondii local isolate into pET-32a(+) and transformed to E. coli BL21.
Highlights
Microneme protein (MIC) is one of proteins that belongs to excretory-secretory antigens (ESAs) of Toxoplasma gondii
Microneme 3 protein (MIC-3) is the protein that plays an important role in the invasion proccess during cell infection as a mediator attachment parasite to the host cell
Deoxyribonucleic acid (DNA) from T. gondii tachyzoites was amplified by PuRe Taq RTG-PCR Beads using mic3 specific primers
Summary
Et al (2003; 2005), dan Buffolano, et al (2005). Protein mikronema (MIC-3) termasuk dalam antigen ekskretori sekretori T. gondii (ESA) yang diduga merupakan kandidat vaksin yang poten dan efektif terhadap toxoplasmosis, karena berperan dalam proses adhesi T. gondii pada sel hospes (Ismael, et al, 2003; Beghetto, et al., 2005; Buffolano, et al, 2005). Pencampuran komponen reaksi untuk proses amplifikasi dilakukan dengan penambahan komponen-komponen ke dalam puRe Taq RTG-PCR sebagai berikut: 2 μl template DNA insert (280 ng/μl), 2 μl primer M3F3 (10 pmol/ μl), dan 2 μl primer M3R3 (10 pmol/ μl) serta. Tabung mikro 0,2 ml yang berisi campuran reaksi tersebut dimasukkan ke dalam thermocycler dan diamplifikasi dengan program sebagai berikut: (1) denaturasi pertama dengan suhu 94 oC selama 5 menit, (2) denaturasi 94 oC selama 1 menit, (3) annealing primer 60 oC selama 1 menit, (4) polimerisasi 72 oC selama 1 menit, (5) siklus diulang sehingga total siklus 35 kali, dan (7) diakhiri dengan polimerisasi tambahan pada suhu 72 oC selama 5 menit. Deoxyribonucleic acid hasil amplifikasi (untuk insert mic3), pWTAM3 (1850 μg/ml) (sebagai kontrol) dari penelitian sebelumnya (Dewi, 2006) dan pET-32a(+) (50 ng/ μl) dipotong menggunakan endonuklease restriksi HindIII dan EcoRV dengan campuran reaksi sebagai berikut: 5 μl DNA, 1 μl enzim HindIII (10 U/μl), 1 μl enzim. Campuran reaksi diinkubasi pada waterbath suhu 37 oC selama 1-2 jam
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