title: Molecular cloning and expression of the cDNA sequence encoding a novel aspartic protease from Uncinaria stenocephala.
abstract: Uncinaria stenocephala belongs to Ancylostomatidae family. Members of this family - hookworms - infect millions of people and animals worldwide. U. stenocephala is most pathogenic in dogs and other Canidae, which are the main hosts, and infection causes anemia or even death. So far no effective hookworm vaccine has been developed that is economically viable. Attempts to identify vaccine antigens have led to a group of aspartic proteases, which play a key role in parasite feeding, migration through host tissues and immune evasion. The cDNA of an aspartic protease from U. stenocephala was cloned using the RACE-PCR method. Computational analysis showed that the cDNA encodes a 447 amino acid protein with a molecular mass of 52kDa that shows high homology to aspartic proteases from related hookworms. Analysis identified 1 potential N-glycosylation site, 3 potential disulfide bonds and no O-glycosylation sites. The recombinant protein was expressed in Escherichia coli followed by purification and mouse immunization. Using raised anti-Us-APR-1(2) (Uncinaria stenocephala Aspartic protease-1) serum the presence of Us-APR-1 in the adult stage of U. stenocephala and the expression of homologous protease in L3 and adult stages of A. ceylanicum was confirmed. This analysis is the first phase of work exploring the biological role of Us-APR-1 in parasite-host interactions and raises hope for successful vaccine development against Uncinaria sp. and possibly Ancylostoma sp.
authors.affiliation: Division of Parasitology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland. katarzyna.wasyl@gmail.com
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Wasyl
authors.forename: Anna
authors.initials: A
authors.lastname: Zawistowska-Deniziak
authors.forename: Piotr
authors.initials: P
authors.lastname: Bąska
authors.forename: Halina
authors.initials: H
authors.lastname: Wędrychowicz
authors.forename: Marcin
authors.initials: M
authors.lastname: Wiśniewski
mesh_terms: D000595:Amino Acid Sequence; D000723:Ancylostomatoidea / Q000201:enzymology* / Q000235:genetics*; D000818:Animals; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D004587:Electrophoresis, Agar Gel; D005243:Feces / Q000469:parasitology; D015971:Gene Expression Regulation, Enzymologic; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008647:Mesocricetus; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D018510:RNA, Helminth / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D018510:RNA, Helminth; D011994:Recombinant Proteins; D057055:Aspartic Acid Proteases