Alarcon-Chaidez Francisco J, Müller-Doblies Uwe U, Wikel Stephen
Center for Microbial Pathogenesis, School of Medicine, University of Connecticut Health Center, Farmington, 06030, USA.
Parasite Immunol. 2003 Feb;25(2):69-77. doi: 10.1046/j.1365-3024.2003.00609.x.
The gene encoding a 36-kDa (p36) immunomodulatory protein present in saliva of Dermacentor andersoni was cloned in prokaryotic and eukaryotic expression vectors. A polymerase chain reaction (PCR)-generated cDNA lacking signal peptide was cloned into the Escherichia coli expression vector pET28 and a similar sequence was cloned into pIB/V5-His-TOPO expression vector for stable transfection of insect cells, High 5 trade mark. The 26-kDa molecular mass of p36 expressed by bacteria is in agreement with that predicted from the translated full-length cDNA sequence. Eukaryotic-cell-expressed p36 consisted of multiple forms with molecular masses between 34 and 36 kDa. These multiple forms were attributed to differences in post-translational modifications. N-linked mannose was detected on insect-cell-expressed and tick-derived p36. Multiple bands remained after endoglycosidase removal of N-linked sugars, indicating the presence of other modifications. Both bacterial- and insect-cell-expressed p36 reacted on immunoblots with polyclonal antibodies raised against tick-derived p36. Insect-cell-expressed p36 suppressed T-lymphocyte-mitogen-driven in vitro proliferation of splenocytes from tick-naïve mice in a dose-dependent manner. Bacterial-cell-expressed p36 lacked immunomodulatory activity.
