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Characterization of a recombinant immunomodulatory protein from the salivary glands of Dermacentor andersoni
Author(s) -
AlarconChaidez Francisco J.,
MüllerDoblies Uwe U.,
Wikel Stephen
Publication year - 2003
Publication title -
parasite immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.795
H-Index - 75
eISSN - 1365-3024
pISSN - 0141-9838
DOI - 10.1046/j.1365-3024.2003.00609.x
Subject(s) - biology , microbiology and biotechnology , complementary dna , polyclonal antibodies , recombinant dna , salivary gland , signal peptide , escherichia coli , antibody , gene , immunology , biochemistry
SUMMARY 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 ™ . 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.