GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 43Gene Ther Mol Biol Vol 7, 43-59, 2003Characterization of genes transcribed in an Ixodesscapularis cell line that were identified by expressionlibrary immunization and analysis of expressedsequence tagsResearch ArticleConsuelo Almazán, Katherine M. Kocan, Douglas K. Bergman, Jose C. Garcia-Garcia, Edmour F. Blouin and José de la Fuente*Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK74078.__________________________________________________________________________________*Correspondence: José de la Fuente, Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma StateUniversity, Stillwater, OK 74078; Phone: (405) 744-0372; Fax: (405) 744-5275; e-mail: jose_delafuente@yahoo.comKey words: tick, vaccine, tick cell culture, cDNA library immunization, EST, expression library immunizationReceived: 23 May 2003; Accepted: 06 June 2003; electronically published: June 2003SummaryExpression library immunization (ELI) combined with analysis of expressed sequence tags (ESTs) were used toidentify genes transcribed in a cell line (IDE8) that was originally derived from embryos of Ixodes scapularis. AcDNA expression library was constructed from the IDE8 cells and cDNA clones were screened by ELI. Miceinjected with cDNA clones were then infested with I. scapularis larvae. cDNA clones affecting larval feeding ordevelopment were subjected to single pass 5’ sequence analysis and the non-redundant sequences were putativelyidentified 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 thataffected larval feeding and/or development were identified, of which 316 cDNA clones contained non-redundantsequences and 101 produced a significant identity to sequences reported previously. Gene ontologies could beassigned to 87 clones. Vaccination of mice with plasmid DNA followed by tick infestation resulted in identificationof cDNA clones that inhibited tick infestation or promoted tick feeding. cDNAs that inhibited tick infestation wereidentical to nucleotidase, heat shock proteins, beta-adaptin, chloride channel, ribosomal proteins, and proteinswith unknown function. cDNA clones that promoted tick feeding were identical to beta-amyloid precursor, block ofproliferation, 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/ordevelopment. These proteins may be useful for incorporation into vaccine preparations designed to interrupt thelife cycle of I. scapularis and/or interfere with transmission of pathogens.I. IntroductionTicks are ectoparasites of wild and domestic animalsand humans, and are considered to be the most importantvector of pathogens in North America (Parola and Raoult,2001). Ixodes spp. (Acari: Ixodidae) are distributedworldwide and are vectors of human pathogens, includingBorrelia burgdorferi (Lyme disease), Anaplasmaphagocytophilum (human granulocytic ehrlichiosis),Coxiella burnetti (Q fever), Francisella tularensis(tularemia), B. afzelii, B. lusitaniae, B. valaisiana and B.garinii, Rickettsia helvetica, R. japonica and R. australis,Babesia divergens, as well as tick-borne encephalitis(TBE) and Omsk Hemorrhagic fever viruses (Estrada-Peña and Jongejan, 1999; Parola and Raoult, 2001).Throughout eastern and southeastern United States andCanada, I. scapularis (the black legged tick) is the mainvector of B. burgdorferi sensu stricto and A.phagocytophilum (Estrada-Peña and Jongejan, 1999;Parola and Raoult, 2001).Control of tick infestations is difficult, particularlyfor multi-host ticks such as Ixodes spp. Presently, tick43