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oai:repository.nwu.ac.za:10394/414652023-05-23T01:08:12Zcom_10394_26463col_10394_26478

Pretorius, P.J. Steyn, Stefanus Johannes 10176705 - Pretorius, Petrus Jacobus (Supervisor) 2023-05-22T09:09:53Z 2023-05-22T09:09:53Z 1995 http://hdl.handle.net/10394/41465 MSc (Biochemie), North-West University, Potchefstroom Campus CLONING AND EXPRESSION OF THE HUMAN GENE FOR INTERLEUKIN-3 (IL-3) The hematopo"ietic growth factor, IL-3, is responsible for the proliferation and differentiation of pluripotent stemcells to mature effector cells in the body. For the formation of mature effector cells, other hematopo"ietic factors also play a role. The key role of IL-3 during hematopo"iesis render this factor great therapeutic potential especially when IL-3 is used in combination with other hematopo"ietic factors. IL-3 is especially successful in the treatment of chemo- and radio-therapy induced secondary hematopo"ietic failure. The genes for GMCSF, G-CSF and IL-5 have already been successful cloned and expressed in our laboratory. As IL-3 has great therapeutic potential and because of the dependance of the above mentioned factors on IL-3 for greater therapeutic success we decided to clone and expressed the human gene for IL-3 Initially we attempt to isolate the IL-3 gene from cDNA and genomic libraries. For this IL-3 specific oligonucleotides were designed and synthesized to be used as probes for the screening of libraries as well as primers during PCR for the amplification of parts of the IL-3 gene. The amplification products can also be used as probes for the screening of libraries. The oligonucleotides were unsuccessful as probes for the isolation of IL-3 cDNAfrom a cDNA library. The use of oligonucleotides as primers for PCR however was successful and two PCR-products were obtained. The longest PCR-product (500bp) was then used to screen a genomic library. All the isolated potential positive clones each time contained only part of the IL-3 fragment. By means of a synthetic IL-3 cDNA probe it was confirmed that all the isolated clones only contained the third, fourth and fifth exon of the IL-3 gene. The synthetic IL-3 cDNA was expressed in an eukaryotic and a prokaryotic expression system. The IL-3 cDNA was successfully cloned in an.eukaryotic expression vector and IL-3 specific biological activity was shown by a recombinant clone. The IL-3 cDNA was also successfully cloned in an prokaryotic expression vector. Two transformed E.coli cell lines produced unique proteins which correspond with the human IL-3 protein. By means of an IL- 3 protein standard and HPLC-analysis it was found that the unique proteins were indeed the human IL-3 protein. Thus the human gene for IL-3 was successfully cloned and expressed in two expression systems. Masters other North-West University (South Africa) Klonering en uitdrukking van die mensgeen vir interleukin-3 (IL-3) Thesis open.access