IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF SIALIDASE (NEURAMINIDASE) GENE AND ENZYME FROM Listeria monocytogenes

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dc.contributor.authorOKERE, Christiana
dc.date.accessioned2015-03-06T13:46:03Z
dc.date.available2015-03-06T13:46:03Z
dc.date.issued2014-08
dc.descriptionA THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES, AHMADU BELLO UNIVERSITY ZARIA NIGERIA IN PARTIAL FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCE AHMADU BELLO UNIVERSITY, ZARIA AUGUST, 2014en_US
dc.description.abstractA putative Trans-sialidase gene was identified in Listeria monocytogenes, a human and animal pathogen and its product was characterized. The identification was carried out using Basic Local Alignment Search Tool (BLAST), Polymerase Chain Reaction (PCR), sequencing and gene analysis/predictions. The enzyme was partially purified from the supernatant of the cultured organism by ammonium sulphate precipitation and gel filtration, the enzyme eluted as a single peak with an activity of 1.6 μM /min. The enzyme was active between pH 4.0 and 4.5, a 45.8% loss of enzyme activity was found at pH 10.0. and an optimum temperature of 30°C, activity gradually decreased with increasing temperature which was reduced to 26% at 70°C and was almost completely lost at 80°C. Effect of cations on the enzyme activity showed that Hg2+ and Al3+ at 3mM concentration are potent inhibitors while Mn2+, Zn2+ and Ca2+ have no effect on the enzyme activity at same concentration compared to the control. The release of the enzyme from the Listeria monocytogenes was time dependent, increasing to 44 × 10-3μM within 60 minutes of incubation. The Trans-sialidase gene was amplified and ligated into pJET1.2 blunt cloning vector to obtain a recombinant DNA which was used to transform E.coli DH5α. The transformed DH5α were selected, the recombinant DNA re-extracted was digested with HindIII and subcloned into pET-28b (+). The sequence analysis revealed a 342bp encoding a 114 translated amino acids protein with a calculated molecular mass of 12.132 kDa and isoelectric point of 8.4. A BLASTP search versus a non-redundant protein sequence database revealed that this protein shows the highest sequence identity to TS2 gene of Trypanosome congolense (100%). 59.6% for T. vivax, T. brucei, and T. evansi, while T.cruzi showed 50% and Sialidase gene of T. rangeli 55.1%. All together, these results suggest that Listeria monocytogenes Trans sialidase might be an important virulence factor and may be involved in the pathogenesis of this organismen_US
dc.identifier.urihttp://hdl.handle.net/123456789/6128
dc.language.isoenen_US
dc.subjectIDENTIFICATION,en_US
dc.subjectMOLECULAR,en_US
dc.subjectSIALIDASE,en_US
dc.subjectNEURAMINIDASE,en_US
dc.subjectENZYME,en_US
dc.subjectListeria,en_US
dc.subjectmonocytogenesen_US
dc.titleIDENTIFICATION AND MOLECULAR CHARACTERIZATION OF SIALIDASE (NEURAMINIDASE) GENE AND ENZYME FROM Listeria monocytogenesen_US
dc.typeThesisen_US
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