IMMUNO-MOLECULAR STUDIES ON BABESI0SIS

Abstract

Babesia microti is a tick-borne hemo protozoan parasite causing murine and human babesiosis. Cysteine Proteases (CPs) are endopeptidases widely distributed in nature and have been identified in a number of protozoan parasites. We identified two different CP genes in B. microti genome (CP1 and CP2), one of them single is copy while the other is multiple copies.

The B. microti parasite extracts showed major CP activity correspond to 22Kda L Mr protoyoltic band on the SOS-PAGE copolymerised with gelatin substrate, this was inhibited by using E-64 (specific CP inhibitor). A synthetic peptide based on a B. microti CP1 homology PCR obtained sequence was used to raise antiserum in rabbits. The rabbit antiserum recognized one putative CP contained in the B. microti parasite extract with apparent 29Kda Mr. The 29Kda CP detected by the anti serum was likely the 22Kda protein with CP activity on gelatin substrate gel. Murine sera with known antibody activities against B. microti slightly reacted with 29Kda antigen, suggesting that this antigen is immune dominant.

Two B. microti CP genes sequence CP1 and CP2 were obtained using 5• and 3'-RACE by gene specific primers derived from the CP1 and CP2 sequence. The mature enzyme region of CP1 sequence has 218 amino acids and the predicted molecular mass is 23Kda while the mature enzyme region of CP2 has 219 amino acids and the predicted molecular mass is 23.2Kda. Both sequence are papain-like and have some homology with