Bekele., Endashaw (Associate Professor)Kidane, Dawit2022-01-112023-11-082022-01-112023-11-081999-06http://etd.aau.edu.et/handle/123456789/29487Molecular 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.enConstruction of Mycobacterial Expression vectorsThesis