Synthesizing and Cloning of Human Immunodeficiency Virus (HIV) and Murine Leukemia Virus (MLV) IRES genes into pBluescript KS(-) Plasmids

E. Ika Prawahju , Silvia Tri Widyaningtyas, Sylvia Sance, Budiman Bela, R. Fera Ibrahim

Research output: Contribution to journalArticlepeer-review

Abstract

In general, translation process of most eukaryotics mRNA occurred for a kind of protein using the 5’ end of cap structure which is a non translated region (NTR). In certain condition, eukaryotics can perform the translation process through internal ribosomal entry site (IRES) as an internal initiation. The mRNAs of some viruses, such as HIV or MLV, perform the process of protein translation without involving NTR, but they are driven by IRES. Therefore, IRES can be applicated to obtained bicistronic mRNA for translation of two different proteins. The goal of the current work is to synthesize and clone DNA fragments containing the IRES genes of Human Immunodeficiency Virus (HIV) and Murine Leukemia Virus (MLV) genes to be used in construction of plasmid DNA for expression of mRNA containing nucleotide sequence of the Avian Influenza H5N1 haemagglutinin (HA) glycoprotein and neuraminidase (NA). The HIV IRES was obtained by PCR using specific primers and the pNL 4.3 plasmid as template. The MLV IRES DNA was generated using mutagenesis PCR method to remove the glicogag initiation codon in the MLV IRES, using pAMS plasmid as template. The removal of initiation codon was intended for the translation process of the glycoprotein to start from the initiation codons of the HA and NA genes. Designed primers for amplification of the HIV IRES and MLV IRES were successfully synthesized to obtain the fragments of respectively 280 bp and 410 bp. After purification, both HIV IRES and MLV IRES fragments were cloned into the pBluescript KS(-) vector separately. Recombinant plasmids containing the HIV IRES and MLV IRES DNA fragments were verified by PCR and enzyme restriction analysis. The DNA fragments containing the HIV IRES and the MLV IRES sequences have been synthesized and cloned into plasmid pBluescript KS(-). Sequencing results of the HIV IRES and MLV IRES fragments showed 98.7% and 99.4% respectively homologous with the HIV IRES-pNL43 plasmid and MLV IRES-pAMS plasmid
Original languageEnglish
JournalJournal of the Indonesian Medical Association
Volume60
Issue number7
Publication statusPublished - 7 Jul 2010

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