Research Project
3500726
Two angiotropins for human endothelial cells have been purified and sequence of the first twenty N-terminal amino acids determined. Only very small amounts of homogenous angiotropins are available from bovine brain and commercial production by conventional means is not economic. Analytical tools (antibody and cDNA) are necessary to assess levels of angiotropins in physiological tissues and fluids in health and disease. Wide availability of angiotropins to the academic and pharmaceutical communities is necessary to understand their chemistry and biology. Availability of angiotropins for routine culture of human endothelial cells from biopsy for diagnostic and prosthetic blood vessels is needed. Long term objectives are to: 1) produce active angiotropins at a commercial scale in recombinant bacteria for use by the academic and pharmaceutical communities; 2) develop diagnostic kits based on antibody or cDNA to determine angiotropin status in fluids and tissues; 3) use inactive analogues of angiotropins or other inhibitors to block their activity in abnormal vascularization. Specific aims of this Phase I study are to: 1) prepare and test polyclonal antibody to synthetic amino acid sequences of angiotropins. Test antibody for use in immunoassay; 2) identify clones of E. coli containing angiotropin cDNA from appropriate cDNA libraries; and 3) assess expression of angiotropin antigen in recombinant clones. Synthetic peptides will be attached to a carrier protein and injected in rabbits to produce antisera. cDNA expression libraries will be prepared using the phage vector, Gammagtll. Recombinant bacterial clones will be identified by synthetic oligonucleotides complementary to N-terminal amino acid sequences of angiotropins.
