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

Urocortin is a recently described 40-meric neuropeptide, which was original ly detected in the rat mid-brain and is believed to play a key role in resp onse 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 expres sed and specific antibodies were generated. So far, the immunological detec tion of urocortin in the rat was largely dependent on antisera generated in rabbits. However, the polyclonal nature of the serum and the remote specie s origin tend to show cross-reactivities and higher backgrounds. On the oth er 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 immuniz ation with a combination of various recombinantly expressed urocortin fusio n proteins resulted in the successful generation of mouse anti-urocortin an tisera, whose specificities were confirmed by ELISA and Western blot analys is. To produce the recombinant proteins for immunization, a cDNA encoding t he mature urocortin sequence was cloned and expressed in fusion either with the glutathione-S-transferase, the maltose-binding protein, thioredoxin, o r a 6X His tag. Depending on the expression system, the solubility and yiel d of the recombinant proteins greatly varied. Together with the newly gener ated antibodies, these recombinantly expressed urocortin proteins will serv e as valuable tools in further investigations of the biological function of urocortin.