Browsing by Author "Kidane, Dawit"

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    Construction of Mycobacterial Expression Vectors
    (Addis Ababa,University, 1999-06) Kidane, Dawit; Bekele., Endashaw (Associate Professor)
    Molecular biology studies on M. feprae and M. tllberculosis involve cloning of genes and high level of expression to provide large amounts of protein. These proteins are used 10 study their usefulness in sub-unit vacci nes or as antigens in diagnostic kits. Most of the work that has been done to obtain protein antigens directly from the pathogens has drawbacks. For example the tedious purification methods and the inability to culture M.leprae in vitro. Most of these obstacles can be circumvented by over-expressing proteins in E.coli but that has its drawbacks as well. The posHransiationai modification in £.coli is absent or at least different from that m mycobacteria. Furthcnnore, some mycobacterial proteins can not be produced in E.coli. We have init iated studies to develop a mycobacterial expression vector th at might sidestep somc or the above namcd difficulties. This study describes the construction or a vector that allows the overexpression or mycobacterial proteins in a non-pathogenic, fast growing mycobacterial host. Firstly, we have cloned a number of model proteins in an E.coli expression system. These vectors allow the over-expression of M.leprae and M.lllberclIlosis recombinant proteins (45kD, Esat-6 and Trx) in E. coli. Secondly, a mycobacteri al expression vector (pDKl) was constructed. This vector has a hi stidine-tag that can be used for affinity purification or proteins. In this way we circumvent tedious biochemical purification systems. It also contains a multiple cloning site for convenient cloning of genes or interest. Thirdly, this pDK I expression vector was used to introduce the same test proteins as for the E.coli expression vector. These test proteins were over-expressed in E. coli as well as in a mycobacterial host. Both protein sets can be used to detcnnine whether there is any difference in recombinant prote ins obtained from E.coli and the semiautologous system using M. smegmaris as a recombinant host.
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    Garlic and orange plant materials evaluation for the control of Sitophilus spp. (Coleoptera: Curculionidae) and Zabrotes subfasciatus (Coleoptera: Bruchidae) in Ethiopia.
    (Addis Ababa University, 2005-06) Kidane, Dawit; Jembere, Bekele (PhD)
    At present much emphasis is being placed in the use of botanical pesticide in the protection of storage insect pests, which have gradually been ignored and also to avoid problems with insecticide resistance. Experiments were conducted to determine the efficacy of Allium sativum L. and Citrus sinesis L. against Sitophilus spp. and Zabrotes subfasciatus under laboratory conditions. For comparison pirimiphos-methyl and untreated check were included and the experiments arranged in a completely randomized design with three replications. One hundred percent mortality of Sitophilus spp. was obtained with water and acetone extracts at the rate of 30g/100ml applied at 3ml/ filter paper after 48 and 96 hour of exposure, respectively. Similar result was obtained for Z. subfasciatus at 20g/100ml rate of extraction. Essential oil from C. sinesis at the highest rate of 750mg/10 ml of acetone applied at 3ml per filter paper gave 100% mortality for both Z. subfasciatus and Sitophilus spp. Allium sativum applied as powder and extracts to the haricot bean were toxic to Z. subfasciatus which caused significant percent mortality of weevils. The highest concentration of acetone extract of A. sativum applied at the rate 15ml/250g of grain caused 82 percent mortality after 96 hours. Grains treated with 15g of sun dried powder of orange peel and 750mg of essential oil killed 65 and 67 percent of Z. subfasciatus after 96 hours respectively. Similarly, A. sativum power had low effect on progeny production of Z. subfasciatus, but grains treated with ethanol and acetone extracts of A. sativum, orange peel powder and essential oil of orange peel reduced progeny production of Z. subfasciatus by more than 92 percent. Citrus sinesis peel oil reduced 100 percent progeny production of Z. subfasciatus at all dosage levels used. All the treatments were repellent to Z. subfasciatus, with the highest dosage of ethanol extracts of A. sativum and fresh chopped garlic evoking the highest repellent action. The treatments, however, showed lower repellency effect against Sitophilus spp. except fresh chopped garlic with 72 percent repellency. The essential oil of orange peel had a high level of toxicity in the fumigation bioassay against Z. subfasiatus and Sitophilus species in impregnated filter paper. The effect of these promising botanicals on weevil progeny survival as well as on seed germination and seed weight loss are discussed.