Characterization of a recombinant immunomodulatory protein from the salivary glands of Dermacentor andersoni.
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.
Alarcon-Chaidez
Francisco J
FJ
Center for Microbial Pathogenesis, School of Medicine, University of Connecticut Health Center, Farmington, 06030, USA.
Müller-Doblies
Uwe U
UU
Wikel
Stephen
S
D000818:Animals; D003001:Cloning, Molecular; D003870:Dermacentor; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation; D015151:Immunoblotting; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D051379:Mice; D011134:Polysaccharides / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012469:Salivary Glands / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology
D011134:Polysaccharides; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C410105:immunosuppressant protein p36, Dermacentor andersoni