In recent years gene therapy has become a promising way of alleviating incurable human ailments, its concept emerging as the ultimate therapy for many infectious and genetic diseases. Two important aspects of the development of successful gene therapy protocols are the ability to monitor gene transfer readily, and the establishment of new protocols for treating specific diseases. In this work, Renilla luciferase and human glutamic acid decarboxylase (GAD) 65 were engineered for secretion to address some aspects of these issues.
Secreted reporter proteins are promising tools to study gene transfer and expression in a non-destructive manner, and bioluminescent proteins are particularly convenient to use for that purpose. To generate a secreted bioluminescent marker protein, secreted Renilla luciferase (SRUC), was engineered by fusing the human interleukin-2 signal peptide to Renilla luciferase. We further modified the Renilla luciferase gene using site-directed-mutagenesis to obtain a mutant form of Renilla luciferase, SRUC3, with dramatically improved stability. SRUC3 provides a rapid, sensitive, and inexpensive assay that does not require disruption of transfected cells. Data from animal experiments suggested that SRUC3 has the potential to be used as an in vivo marker protein. Although SRUC3 activity was not detected from animal blood, data indicated that improvement of the assay after further engineering of the sruc3 gene and SRUC3 protein could allow detection of Renilla luciferase activity in the blood and serum in the future.
To evaluate the potential of genes encoding engineered secreted autoantigens for the treatment of autoimmune disease using gene vaccination, human GAD65 was engineered to be secreted by mammalian cells. GAD65 is a major autoimmune antigen involved in the development of type I diabetes in both non-obese diabetic (NOD) mice and human patients. After the removal of the N-terminal end of GAD65, the truncated GAD fragment was fused to the C-terminal of the human interleukin-2 leader peptide, and a secreted form of GAD (SGAD55) was obtained. The intramuscular injection of plasmid DNA encoding GAD65 and SGAD55 into three-week-old NOD mice showed a dramatic reduction of development of insulitis, the first symptom of diabetes in NOD mice.
Microbiology and Molecular Genetics
Microbiology, Molecular Biology and Biochemistry
Alan P. Escher
John J. Rossi
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Genetic Engineering; Protein Engineering; Gene Therapy; Gene Transfer; Enzymes, Coenzymes, and Enzyme Inhibitors; Luciferase; Genetic Engineering. Protein Engineering. Gene Therapy. Gene Transfer. Enzymes, Coenzymes, and Enzyme Inhibitors. Luciferase. Vaccines, Synthetic.
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Liu, JingXue, "Engineering Secreted Proteins for Gene Transfer and DNA Vaccination" (1999). Loma Linda University Electronic Theses, Dissertations & Projects. 728.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives