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Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization
Authors:A Mulenga  O Misao  C Sugimoto
Institution:(1) Dept. of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, 21201, USA;(2) Graduate School of Veterinary Medicine, Hokkaido University, Kitaku, Kita 18, Nishi 9, Sapporo, 060-0818, Japan;(3) The National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, 080-8555, Japan
Abstract:Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3prime and 5primerapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His57 and Ser195 conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.
Keywords:Rhipicephalus appendiculatus  Serine proteinases  Tick vaccine
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