AMPLIFIKASI SECARA PCR, KLONING PADA YEKTOR PMOSBLUE, DAN SEKUENSING FRAGMEN DNA 0,6 KILO BASA GEN IVIIIISALMONELLA TYPHIMURIUM

• Main Author: Salirawati, Das
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/4573
• Institution: Institut Teknologi Bandung

<b>Abstract: <p align=\"justify\"> <br /> Salmonella typhi is a pathogenic bacterium causing typhoid fever on man. The molecular pathogenesis mechanism of S. typhi is not well understood. This is especially due to the genes themselves are not known yet. More research works are required to understand the mecanism. A similar species, called Salmonella typhimurium, may cause typhoid disease on mice. The species may be applicable as a model to study the typhoid disease on man since five ivi (in vivo induced) genes were discovered from the species using In Vivo Expression Technology (IVET) method and suspected to be responsible for the molecular pathogenesis mechanism. One of the genes, iviIII gene, suspected homolog with rfbP gene that found in the rfb operon. This research aims to find out the in vivo expressed genes of S. typhi, and especially to obtain the iviIII gene from S. typhimurium using PCR method, followed by nucleotide sequencing of its DNA fragment by the dideoxy-Sanger method. DNA fragment of 0,6 kb of iviIIl gene obtained from S. typhimurium (Mutton) (NCTC 74, ATCC 13311) had been amplified using rfb l and rf b2 primers that were designed based on the nucleotide sequence of S. typhimurium LT2 rfbP gene. Restriction analysis using Sau 3M and EcoR I enzymes comfirmed that the fragment was part of the rfbP gene and the insert of recombinant plasmid was the 0,6 kb DNA. Nucleotides, 565 basepairs, were obtained by the dideoxy-Sanger method from the 0,6 kb DNA fragment of the amplified S. typhimurium iviIII gene which was cloned with pMOSBlue T-vector. The first and the last 20 nucleotides were the rfbl and the rfb2 primers nucleotide sequence respectively, while the remaining 525 nucleotides were showed 100% homology with the nucleotide sequence of the S. typhimurium LT2 rfbP gene. This result demonstrates that the amplified 0,6 kb DNA fragment of the S. typhimurium ivilll gene was part of the S. typhimurium LT2 rfbP gene.<p align=\"justify\">