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Folia Medica Indonesiana

ISSN 0303-7932

Vol. 49 / No. 2 / Published : 2013-04

TOC : 7, and page :101 - 108

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Original Article :

Direct cloning of melanoma antigen 1 (mage-1) and the e2 gene of hog cholera virus (hcv) from the blunt end pcr product using pet 101/d-topo

Author :

  1. Gondo Mastutik*1
  2. Rosmelati Situmeang*2
  1. Dosen Fakultas Kedokteran
  2. Technical Services Unit Area, Veterinary Public Health Laboratory, East Kalimantan

Abstract :

The objective of this study was to clone the full length of coding region of Mage-1 and E2 gene of Hog Cholera Virus (HCV) usingthe plasmid pET101/D-TOPO to get the corresponding recombinant plasmid. Samples were obtained from the liver tissue withhepatocellular carcinoma and a pig spleen infected by HCV from Sukoharjo Middle Java. cDNA were amplified by PCR usingGMTOPOF-GMTOPOR for Mage-1 and RS-1-RS-2 for E2 HCV.The purified PCR products were cloned into pET101/D-TOPO asdirect cloning expression vector. The gene target was transformed into E. coli Top10. Analysis of the recombinant plasmid wasundertaken by sequencing and restriction test. The PCR of Mage-1 gene resulted fragments at + 1105bp for first round PCR and at+ 931bp for the second round. The PCR of E2 gene HCV produced fragment at + 1200 bp. The DNA targets were cloned intopET101/D-TOPO from the blunt end PCR product directly. The sequence of full length of coding region of Mage-1 contained 927nucleotides that encoded 309 amino acids residues. The sequence of the full length coding region of E2 gene HCV was encoded by1218 nucleotide. EcoRV enzyme cuts vector pET101/D-TOPO at nucleotide positions at 545 and 4775bp. The result of EcoRVrestriction had band at 4230 and 2450 for pETGM/MAGE1-HCC and 4200 and 2700bp for pETRS/E2-SH. The recombinant plasmidpETGM/MAGE1-HCC and pETRS/E2-SH were obtained from the blunt end PCR product for direct cloning using pET101/D-TOPO.(FMI 2013;49:101-108)

Keyword :

direct cloning, Mage-1, E2 Hog Cholera Virus, pET101/D-TOPO,

References :

  1. Casali N and Preston A, (2003). E. coli Plasmid Vectors: Methods and Applications. Totowa : Humana Press
  2. Mastutik G, Hardjowijoto S, Lunardi JH, Puspaningsih NNT, Soetjipto, Kusumobroto HO, Putra ST, (2008). Molecular analysis of the coding sequence of mage-1 gene fromhepatocelular carcinoma patient and testis. - : Folia Medica Indonesiana


Archive Article

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Volume : 49 / No. : 2 / Pub. : 2013-04
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  2. Mycobacterium tuberculosis immunoglobulin m (igm) and immunoglobulin g (igg) survey in hiv-infected donors
  3. Comparing the effect of red yeast rice, date palm, and guava leaf extract on thrombocyte and megakaryocyte count in thrombocytopenic white rats
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  5. Rapid culture method using biphasic media on bacteriologic diagnosis to detect mycobacterium tuberculosis for determining pulmonary tuberculosis
  6. Effect of carbide and cacl2 concentration on vitamin c contents of banana kepok
  7. Direct cloning of melanoma antigen 1 (mage-1) and the e2 gene of hog cholera virus (hcv) from the blunt end pcr product using pet 101/d-topo
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