The proliferation-associated nuclear protein Ki-67 in the bovine system: partial characterisation and its application for determination of the proliferation of Theileria-infected bovine cells

Theileria annulata-infected bovine cells as well as mitogen-stimulated bovi ne peripheral blood mononuclear cells (PBMC) express a proliferation-associ ated nuclear protein equivalent to the human Ki-67 protein. In analogy to t he human system, the expression of the bovine Ki-67 protein is restricted t o proliferating cells only, since (a) Ki-67 expression paralleled [H-3]-thy midine incorporation in concanavalin A (Con A)-stimulated bovine PBMC, (b) Ki-67 was not detectable in quiescent bovine cells, and (c) Ki-67 expressio n in Theileria-infected cells is related to the presence of the parasites w ithin the cytoplasm of the host cells; upon treatment with the theilericida l drug buparvaquone the parasites are destroyed and the cells cease to prol iferate and to express the Ki-67 protein. Western-blot analysis of lysates of proliferating bovine cells revealed that the prototype monoclonal antibo dy Ki-67 and the new equivalent antibody MIB-1 detected one prominent prote in band with an apparent molecular weight of 430 kDa. Two cDNA clones (pUC1 8.B1.Ki-67 and pUC18.B2.Ki-67) were isolated from a lambda gt11 cDNA librar y of T. annulata-infected bovine cells by immunoscreening with the monoclon al antibody MIB-1. Comparison of these cDNA sequences with those of the hum an Ki-67 protein revealed 60-70% identity. Within the "Ki-67 motif", identi ty proved to be 80% at the amino acid level. The remarkable identity betwee n bovine and human Ki-67 proteins suggests that MIB-1 can be used as a mark er for cell proliferation in animal research. In this context we could iden tify proliferating cells in lymph nodes of Theileria-infected animals and, furthermore, we could distinguish between infected and uninfected prolifera ting cells using MIB-1 and an antiserum against a recombinant parasite prot ein designated SA(288).