Kloning Gen Melanoma Antigen 1 (Mage-1) dari Jaringan Testis untuk mendapatkan Plasmid Rekombinan Mage-1

Gondo Mastutik, Reny I’tishom, Sunaryo Hardjowijoto, Suhartono Taat Putra

Abstract


Gen Melanoma antigen-1 (Mage-1) diekspresikan oleh sel spermatogonia jaringan testis normal dan diekspresikan 60−80% oleh liver penderita karsinoma hepatoseluler (KH). Ekspresi Mage-1 merupakan penanda untuk diagnosis KH serta prediktor kanker lambung dan kolorektal. Isolasi messenger ribonucleid acid (mRNA) Mage-1 dari jaringan liver penderita KH sulit dilakukan sehingga dilakukan isolasi mRNA Mage-1 dari jaringan yang mengekspresikan Mage-1, yaitu jaringan testis normal. Penelitian ini merupakan penelitian eksploratif yang dilakukan di Lembaga Penyakit Tropis Universitas Airlangga, Agustus 2006–Agustus 2008. Tujuan untuk mengkloning seluruh area koding gen Mage-1 dari jaringan testis pada vektor dan mendapatkan plamid rekombinan Mage-1. Isolasi seluruh area koding gen Mage-1 dilakukan dengan teknik semi-nested polymerase chain reaction (PCR). Seluruh area koding gen Mage-1 diisolasi, kemudian dikloningkan ke plasmid pET101/D-TOPO dan ditransformasikan ke Escherichia coli (E. coli) Top10 untuk mendapatkan plasmid rekombinan Mage-1. Panjang pET101/D-TOPO adalah 5.753pb dan area koding gen penyandi Mage-1 927 bp sehingga total panjang plasmid rekombinan 6.680 bp (5.753+927). Hasil analisis restriksi dengan EcoRV menunjukkan pita 4.230 dan 2.450 (4.230+2.450 = 6.680). Analisis sekuens gen Mage-1 dari testis mempunyai homologi 100% dengan sekuens M77481 serta NM_004988, dan 99% dengan BC01755. Simpulan, berdasarkan hasil analisis restriksi dan sekuens maka diperoleh plasmid rekombinan pETGM/MAGE1-Testis yang mengandung seluruh area koding gen Mage-1 dan dapat digunakan untuk pengembangan kit diagnostik karsinoma.  [MKB. 2015;47(4):199–206]

Kata kunci:  Jaringan testis, karsinoma hepatoseluler, kloning, melanoma antigen-1, pET101/D-TOPO

Cloning of Melanoma Antigen 1 (Mage-1) Gene from Testicular Tissue to Obtain the Recombinant Plasmid Mage-1

Melanoma antigen-1 (Mage-1) is expressed by spermatogonia cells of normal testicular tissue and 60−80% is expressed by the liver of hepatocellular carcinoma (HC) patients. Mage-1 expression is a marker for diagnosing HC and predicting gastric and colorectal cancers. Isolation of messenger ribonucleid acid (mRNA) Mage-1 from the liver tissue of HC patients is difficult; therefore, Mage-1 mRNA isolates can be obtained from tissues that express Mage-1 such as normal testicular tissues . This is an explorative research that was conducted at the Institute of Tropical Diseases of Airlangga University during August 2006–August 2008. The aim was to clone the coding sequence of Mage-1 gene from testicular tissues into a vector and to get recombinant plasmid Mage-1. Isolation of the full-length Mage-1 was performed using semi-nested polymerase chain reaction (PCR) which was then cloned into plasmid pET101/D-TOPO and transformed into Escherichia coli (E. coli) Top10 to get recombinant plasmid Mage-1. The length of pET101/D-TOPO was 5,753 bp and Mage-1 was 927 bp. The length of recombinant plasmid was 6,680 bp (5,753+927). Restriction analysis using EcoRV showed 4,230 and 2,450 bp bands (4,230+2,450=6,680). Sequence analyses showed that Mage-1 was 100% homologous with M77481 and NM_004988, 99% homologous with BC01755. In conclusion, according to the results of the restriction and sequences analysis, the recombinant plasmid pETGM/MAGE1-Testis contains the full length coding region of Mage-1 and is useful for developing the hepatocellular carcinoma diagnostic kits. [MKB. 2015;47(4):199–206]

 

DOI10.15395/mkb.v47n4.621


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