Generation of self-antigen reactive, anti-urocortin specific antibodies by immunization of recombinantly expressed urocortin fusion proteins

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Title
Generation of self-antigen reactive, anti-urocortin specific antibodies by immunization of recombinantly expressed urocortin fusion proteins
Author(s)
Shin Young Na; Jung Hyun Park; Eun Wie Cho; Kwan Hee You; Kil Lyong Kim
Bibliographic Citation
Molecules and Cells, vol. 9, no. 6, pp. 587-595
Publication Year
1999
Abstract
Urocortin is a recently described 40-meric neuropeptide, which was originally detected in the rat mid-brain and is believed to play a key role in response to stress situations. While its function in the central nervous system is rather well established, the biological role in the periphery is still to be determined. To investigate its distribution and effect on peripheral cells and tissues, in the present study, urocortin was recombinantly expressed and specific antibodies were generated. So far, the immunological detection of urocortin in the rat was largely dependent on antisera generated in rabbits. However, the polyclonal nature of the serum and the remote species origin tend to show cross-reactivities and higher backgrounds. On the other hand, generation of mouse antibodies to rat urocortin was hampered since mouse and rat urocortin sequences are identical, and such antibodies would represent auto-reactive antibodies. Despite such restrictions, the immunization with a combination of various recombinantly expressed urocortin fusion proteins resulted in the successful generation of mouse anti-urocortin antisera, whose specificities were confirmed by ELISA and Western blot analysis. To produce the recombinant proteins for immunization, a cDNA encoding the mature urocortin sequence was cloned and expressed in fusion either with the glutathione-S-transferase, the maltose-binding protein, thioredoxin, or a 6X His tag. Depending on the expression system, the solubility and yield of the recombinant proteins greatly varied. Together with the newly generated antibodies, these recombinantly expressed urocortin proteins will serve as valuable tools in further investigations of the biological function of urocortin.
Keyword
6X HistidineAntibodiesGlutathione S-transferaseMaltose Binding ProteinThioredoxinUrocortin
ISSN
1016-8478
Publisher
Korea Soc-Assoc-Inst
Type
Article
Appears in Collections:
Division of Biomedical Research > Rare Disease Research Center > 1. Journal Articles
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