Cloning and Sequence Analysis of a cDNA Encoding Pso o II, a Mite Group II Allergen of the Sheep Scab Mite (Acari: Psoroptidae)

• Published in: Journal of medical entomology 2002-03, Vol.39 (2), p.384-391
• Main Authors: Temeyer, Kevin B., Carmen Soileau, L., Pruett, John H.
• Format: Article
• Language: English
• Subjects: Allergens; Allergens - classification; Allergens - genetics; Allergy; Amino Acid Sequence; amino acid sequences; Animals; Base Sequence; Biological and medical sciences; Cloning; Cloning, Molecular; complementary DNA; DNA, Complementary; Fundamental and applied biological sciences. Psychology; Genomic libraries; Glycine; Leptin; Medically important nuisances and vectors, pests of stored products and materials: population survey and control; mite allergens; mite group II allergens; Mites - genetics; Molecular Sequence Data; nucleotide sequences; Nuisances; Phylogeny; Psoroptes ovis; psoroptic scabies; scabies; Sequence Analysis, DNA; Sheep - parasitology; sheep scab mite
• ISSN: 0022-2585; 1938-2928
• DOI: 10.1603/0022-2585-39.2.384

Psoroptes ovis (Hering), the sheep scab mite, is responsible for psoroptic scabies of cattle and sheep. Reverse translation of 30 N-terminal amino acids of the major P. ovis allergen, previously chosen as a candidate immunogen and identified as a 16 kDa protein yielded a degenerate sequence used to design oligodeoxynucleotide polymerase chain reaction (PCR) primers. Use of the PCR primers with a P. ovis cDNA library succeeded in amplification of a 90 bp cDNA gene fragment that was cloned, sequenced, and used to select unique sequencing/PCR primers. Primer walking generated overlapping subclones which yielded the 588 nucleotide consensus sequence of the cDNA encoding the 143 amino acid P. ovis allergen precursor. Nucleotide and translated sequences of the cDNA were compared with sequences in GenBank and found to be homologous to mite group II allergens Lep d II (formerly Lep d I) of Lepidoglyphus destructor Schrank, Der f II of Dermatophagoides farinae Hughes, Der p II of Dermatophagoides pteronyssinus (Trouessart), Tyr p II of Tyrophagus putrescentiae (Schrank), Eur m II of Euroglyphus maynei (Cooreman) and Gly d II of Glycophagus domesticus (De Geer). The mature P. ovis allergen is composed of 126 amino acids with a calculated molecular mass of 13,468 Da, three disulfide bonds, and pI of 6.06 with one potential o-glycosylation site at Thr116. We designate the P. ovis 16 kDa protein as Pso o II in conformity with nomenclature for mite group II allergens. RESUMEN Psoroptes ovis (Hering), el ácaro de la sarna de los ovinos, es responsable de la sarna psoróptica del ganado y las ovejas. Traducción reversa de 30 amino ácidos de la región N-terminal del alérgeno mayor de P. ovis, fue previamente escogido como un candidato a immunógeno e identificado como una proteína de 16 kDa, de la cual se diseñaron oligonucleótidos iniciadores degenerados para PCR. El uso de los iniciadores con una geneteca de cDNA de P. ovis, fueron exitosos para amplificar un fragmentode cDNA de 90 pares de bases, los cuales fueron clonados, secuenciados y usados para seleccionar iniciadores específicos para PCR. Iniciadores detraslape de secuencias generaron subclonas, las cuales resultaron en una secuencia consenso de cDNA de 588 nucleótidos, que codifica el alergeno precursor de 143 amino ácidos de P. ovis. Las secuencias traducidas de nucleótidos de cDNA fueron comparadas con secuencias en el GenBank y se encontraron homólogas al grupo II de alergenos de ácaros Lep d II (antes Lep d I)