Gene Therapy and Molecular Biology Vol 7, page 43 Gene Ther Mol Biol Vol 7, 43-59, 2003
Characterization of genes transcribed in an Ixodes scapularis cell line that were identified by expression library immunization and analysis of expressed sequence tags Research Article
Consuelo Almazán, Katherine M. Kocan, Douglas K. Bergman, Jose C. GarciaGarcia, Edmour F. Blouin and José de la Fuente* Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
__________________________________________________________________________________ *Correspondence: José de la Fuente, Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078; Phone: (405) 744-0372; Fax: (405) 744-5275; e-mail: jose_delafuente@yahoo.com Key words: tick, vaccine, tick cell culture, cDNA library immunization, EST, expression library immunization Received: 23 May 2003; Accepted: 06 June 2003; electronically published: June 2003
Summary Expression library immunization (ELI) combined with analysis of expressed sequence tags (ESTs) were used to identify genes transcribed in a cell line (IDE8) that was originally derived from embryos of Ixodes scapularis. A cDNA expression library was constructed from the IDE8 cells and cDNA clones were screened by ELI. Mice injected with cDNA clones were then infested with I. scapularis larvae. cDNA clones affecting larval feeding or development were subjected to single pass 5’ sequence analysis and the non-redundant sequences were putatively identified by sequence identity using the protein Basic Local Alignment Search Tool (BLAST) algorithm. Sequences of the clones were grouped according to the predicted function of the encoded proteins. 351 cDNAs that affected larval feeding and/or development were identified, of which 316 cDNA clones contained non-redundant sequences and 101 produced a significant identity to sequences reported previously. Gene ontologies could be assigned to 87 clones. Vaccination of mice with plasmid DNA followed by tick infestation resulted in identification of cDNA clones that inhibited tick infestation or promoted tick feeding. cDNAs that inhibited tick infestation were identical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins, and proteins with unknown function. cDNA clones that promoted tick feeding were identical to beta-amyloid precursor, block of proliferation, mannose-binding lectin, RNA polymerase III, ATPases and a protein of unknown function. Herein, we describe the sequence analysis of I. scapularis ESTs selected by ELI that affected larval tick feeding and/or development. These proteins may be useful for incorporation into vaccine preparations designed to interrupt the life cycle of I. scapularis and/or interfere with transmission of pathogens. garinii, Rickettsia helvetica, R. japonica and R. australis, Babesia divergens, as well as tick-borne encephalitis (TBE) and Omsk Hemorrhagic fever viruses (EstradaPeña and Jongejan, 1999; Parola and Raoult, 2001). Throughout eastern and southeastern United States and Canada, I. scapularis (the black legged tick) is the main vector of B. burgdorferi sensu stricto and A. phagocytophilum (Estrada-Peña and Jongejan, 1999; Parola and Raoult, 2001). Control of tick infestations is difficult, particularly for multi-host ticks such as Ixodes spp. Presently, tick
I. Introduction Ticks are ectoparasites of wild and domestic animals and humans, and are considered to be the most important vector of pathogens in North America (Parola and Raoult, 2001). Ixodes spp. (Acari: Ixodidae) are distributed worldwide and are vectors of human pathogens, including Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic ehrlichiosis), Coxiella burnetti (Q fever), Francisella tularensis (tularemia), B. afzelii, B. lusitaniae, B. valaisiana and B.
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