Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Kenneth L. White

Second Advisor

Donald L. Thompson, Jr

Abstract

Pathogens continue to evolve mechanisms of avoiding efforts to control their spread. Treatment with exogenous antimicrobials often occurs after the pathogen has reproduced extensively in the host. In the case of mammals, the immune system may not be fully mobilized for 72 h after the detection of foreign antigen. However, within 24 h of presentation of antigen by antigen-presenting cells to resting thymus-derived lymphocytes IL2 is produced. A class of broad spectrum antimicrobial peptides which constitute a major component of the insect immune system has been described and characterized. Called "cecropins", they are relatively non-toxic to normal cells from multicellular organisms but are toxic to a wide range of bacteria, protozoa, and fungi as well as infected and abnormal cells. The experiments presented here describe the construction of a plasmid vector and transcription of mRNA for a cecropin-class synthetic lytic peptide, Shiva 1, under control of the mouse IL2 promoter/enhancer and linked to the IL2 signal sequence. Twenty-eight lines of transgenic mice were produced by pronuclear injection of a BamHI fragment containing from $-$593 to +110 of the 5$\sp\prime$ flanking region of mouse interleukin 2 gene including the peptide signal sequence, Shiva 1, and the SV40 polyadenylation signal and splice site. By a reverse-transcription polymerase chain reaction (RT-PCR) assay, it was determined that two lines of transgenic mice were produced whose spleen derived lymphocytes could be induced to transcribe and mature mRNA for Shiva 1 by exposure to 3.25 $\mu$g/ml of concanavalin A. In addition, one of the lines showed evidence of in vivo expression. Since the controlling regions of the IL2 enhancer and amino acid sequence of the signal peptide are highly conserved among humans, mice, cattle, and sheep, it is likely that this recombinant gene will function in a variety of mammals. Studies to assess immune system enhancement of the mice are ongoing.

Pages

69

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