GTMB 7 - Gene Therapy & Molecular Biology

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Almazán et al: Expressed sequence tags in Ixodes scapulariscontrol is effected by integrated pest management inwhich different control methods are adapted in ageographic area against one tick species with dueconsideration to their environmental effects. Recently,development of vaccines against one-host Boophilus spp.has provided new possibilities for identification ofprotective antigens for use in vaccines for control of tickinfestations (Willadsen, 1997; Willadsen and Jongejan,1999; de la Fuente et al, 1999, 2000a; de Vos et al, 2001).Control of ticks by vaccination would avoid environmentalcontamination and selection of drug resistant ticks that canresult from repeated acaricide application (de la Fuente etal, 1998; Garcia-Garcia et al, 1999). Anti-tick vaccinesalso allow for inclusion of multiple antigens in order totarget a broad range of tick species, as well as pathogenblockingantigens.Development of high throughput DNA sequencingtechnologies and bioinformatic tools facilitate assignmentof provisional function to expressed sequence tags (ESTs;Boguski et al, 1993). This approach has resulted invaluable information for the study of biological systemsand for the identification of potential vaccine candidates(Lizotte-Waniewski et al, 2000; Knox et al, 2001; Tarletonand Kissinger, 2001; Touloukian et al, 2001; Kessler et al,2002). In ticks, construction of EST databases has beenreported for B. microplus (Crampton et al, 1998),Amblyomma americanum (Hill and Gutierrez, 2000) andA. variegatum (Nene et al, 2002). The application of ESTtechnology has been used for characterization of geneexpression in salivary glands of I. scapularis (Valenzuelaet al, 2002), I. ricinus (Valenzuela, 2002), A. americanumand Dermacentor andersoni (Bior et al, 2002), foridentification of genes differentially expressed in D.variabilis ovaries in response to rickettsial infection(Mulenga et al, 2003) and in I. ricinus salivary glands inresponse to blood feeding (Leboulle et al, 2002).A new technique, expression library immunization(ELI), in combination with sequence analysis of ESTs,provides an alternative approach for identification ofpotential vaccine antigens that is based on rapid screeningof the expressed genes without prior knowledge of theantigens encoded by the cDNAs. ELI was first reported forMycoplasma pulmonis (Barry et al, 1995) and since thenhas been used for unicellular and multicellular pathogensand viruses (Manoutcharian et al, 1998; Alberti et al,1998; Brayton et al, 1998; Melby et al, 2000; Smooker etal, 2000; Moore et al, 2001; Singh et al, 2002; Leclercq etal, 2003). Recently, we reported the first application ofELI to arthropods, specifically to I. scapularis (Almazánet al, 2003) in a mouse model system. A combination ofcDNA ELI and EST analysis resulted in the selection of351 cDNA clones affecting tick larval development(Almazán et al, 2003). After grouping the clonesaccording to the putative function of predicted proteins,some cDNA pools resulted in the inhibition of tickinfestation and others promoted tick feeding after ELI(Almazán et al, 2003).Herein we describe the sequence analysis andcharacterization of I. scapularis ESTs that were identifiedby Almazán et al. (2003) using cDNA ELI and a mousemodel for tick infestation.II. Materials and methodsA. Construction of the I. scapularisexpression cDNA libraryThe cDNA library was constructed from I. scapulariscultured embryonic IDE8 cells (Munderloh et al, 1994) asreported previously (Almazán et al, 2003). The expression librarywas constructed in the vector pEXP1 containing the stronghuman cytomegalovirus major immediate earlypromoter/enhancer (CMV IE ) (Clontech, Palo Alto, CA). ThecDNA library contained 4.4 x 10 6 independent clones and a titerof approximately 10 10 cfu/ml with more than 93% of the cloneswith cDNA inserts. The average cDNA size was 1.7 kb (0.5-4.0kb).B. DNA vaccination and tick infestationVaccinations with plasmid DNA and tick infestations weredone as reported previously for the screening of the expressioncDNA library by ELI using the mouse model of I. scapualrisinfestations (Almazán et al, 2003). Briefly, plasmid DNA waspurified (Wizard SV 96 plasmid DNA purification system,Promega, Madison, WI) and used to inject CD-1 female mice, 5-6 weeks of age at the time of first vaccination. Mice were caredfor in accordance with standards specified in the Guide for Careand Use of Laboratory Animals. Mice were injected using a 1 mltuberculin syringe and a 27αG needle at days 0 and 14. Three to6 mice per group were each immunized IM in the thigh with 1 µgtotal DNA/dose in 50 µl PBS. Control mice were injected with 1µg vector DNA alone. Two weeks after the last immunization,mice were infested with 100 I. scapularis larvae per mouse. Fortick infestations, mice were retrained in a small wire cage in acardboard carton. One hundred larvae were counted and appliedto the mice with a brush. Ticks were reared at the OklahomaState University Tick Rearing Facility by feeding larvae on mice,nymphs on rabbits and adults on sheep. For these experiments,larvae were obtained from the eggs oviposited by sister females.Twelve hours after tick infestation, larvae in the bottom of thecage that did not attach were counted in order to calculate thenumber of attached larvae per mouse. Mice were then transferredto individual cages in which they were placed on an elevated1/4” mesh wire platform over water (1/2” deep). Replete larvaedropping from each mouse were collected daily from the waterand counted during 7 days. Time for larval development wasevaluated from the day of tick infestation to the day in which themaximum number of replete larvae was collected. The inhibitionof tick infestation (I) for each test group was calculated withrespect to vector-immunized controls as [1-(RLn/RLc xRLic/RLin)] x 100, where RLn is the average number of repletelarvae recovered per mouse for each test group, RLc is theaverage number of replete larvae recovered per mouse for controlgroup, RLic is the average number of larvae attached per mousefor control group, and RLin is the average number of larvaeattached per mouse for each test group. Engorged larvae wereheld in a 95% humidity chamber and allowed to molt. Molting ofengorged larvae was evaluated 34 days after the last larvalcollection by visual examination of ticks under a dissecting lightmicroscope. The inhibition of molting (M) for each test groupwas calculated with respect to controls as [1-(MLn/MLc xRLc/RLn)] x 100, where MLn is the average number of nymphsfor each test group, MLc is the average number of nymphs forthe control group, RLc is the average number of larvae recoveredfor the control group, and RLn is the average number of larvaerecovered for each test group.44
Gene Therapy and Molecular Biology Vol 7, page 45C. Plasmid DNA preparation and sequencingBacterial colonies were inoculated in Luria-Bertani with 50µg/ml ampicillin, grown for 16 hr in a 96-well plate and plasmidDNA purified (Wizard SV 96 plasmid DNA purification system,Promega, Madison, WI) and partially sequenced with a 5’ vectorspecificprimer (5’-CGACTCACTATAGGGAG-3’) at the CoreSequencing Facility, Department of Biochemistry and MolecularBiology, Noble Research Center, Oklahoma State University,using ABI Prism dye terminator cycle sequencing protocolsdeveloped by Applied Biosystems (Perkin-Elmer Corp., FosterCity, CA). In most cases a sequence larger than 700 nucleotideswas obtained.D. Data analysisNucleotide sequences were analyzed using the programAlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda,MD). Multiple sequence alignment was performed using anengine based on the Clustal W algorithm (Thompson et al, 1994).Nucleotides were coded as unordered, discrete characters withfive possible character-states; A, C, G, T, or N (missing) andgaps were coded as missing data. Phylogenetic trees wereconstructed based on a sequence distance method utilizing theNeighbor Joining algorithm of Saitou and Nei (1987). BLAST(Altschul et al, 1990) was used to search the NCBI databases toidentify previously reported sequences with identity to those thatwe sequenced. Gene ontology assignments were made accordingto Ashburner et al. (2000) for non-redundant EST sequence datawith the help of GoFish v.1.0 (Berriz et al, 2003).III. ResultsThe screening of the I. scapularis expression cDNAlibrary by ELI and EST analysis resulted in 351 cDNAsaffecting larval development in the mouse model of tickinfestation (Almazán et al, 2003). Of them, 316 cDNAclones contained non-redundant sequences and 101 (32%)produced a significant identity to previously reportedsequences by BLAST analysis of NCBI nucleotide andprotein databases (Table 1). Gene ontologies could beassigned to 87 clones (27.5% of non-redundant sequencesand 86.1% of clones with identity to sequences reportedpreviously) (Table 2).Table 1. cDNA clones with identity to previously reported sequences.EST clone Predicted protein GenBankaccessionnumber1C111E6Translation initiation factor 5A(eIF5A)Translation initiation factor 5C(eIF-5C)CD052489CD0524902D2 Initiate factor 5 (if5) CD0524911A10 Elongation factor 2 CD0524924F7 Elongation factor 1alpha CD0524931F6 Ribosomal protein S4 (RpS4) CD0524942B8 Ribosomal protein S11 (RpS11) NR2F8Laminin receptor 1 (ribosomalprotein SA)CD0524962F10 Ribosomal protein L3 (RpL3) NR3A10 Ribosomal protein L7A (RpL7A) CD0524973G9 Ribosomal protein S8 (RpS8) CD0524953G103C3Ribosomal protein L27A(RpL27A)QM homolog (DQM) ribosomalproteinCD052498CD0524994D12 Proteasome/Signalosome subunit CD0525004E7 Proteasome subunit CD0525014D11 Proteasome subunit CD0525023D10 Ribophorin I CD0525031B12 Ubiquitin-conjugating enzyme CD0525041D10 Ubiquitin CD0525051A9V-ATPase D subunitContains microsatellite sequenceCD0525061B2 V-ATPase C subunit CD052507EST clone Predicted protein GenBankaccessionnumber4A4 V-ATPase E subunit CD0525081C5 Na+/K+ ATPase, alpha subunit CD0525092A9 NADH dehydrogenase CD0525101D6 NADH dehydrogenase subunit 5(nad5)CD0525111A4 Aldehyde dehydrogenase CD0525121C8 Virilizer (vir) CD0525131C10 Hsp70 CD0525143F6 Hsp60 CD0525151D1 Nucleotide binding protein 1(Nubp1)1D8Identity to D. melanogasterGH03607 full length cDNAcoding for a putative membraneproteinCD052516CD0525171D11 Putative membrane protein CD0525181E7 Sterol carrier protein CD0525191F3 Cyclin C (CycC) CD0525203D9 Alpha tubulin CD0525212A7 Beta tubulin CD0525222A11 Notchless (Nle) CD0525232B2 Export factor binding protein 2(Refbp2)CD0525242B7 G protein-coupled receptor CD0525252B92C122D1Succinate dehydrogenase B(SdhB)Beta-amyloid precursor protein(APP)Fructose-1,6-bisphosphatase (fbpgene)CD052526CD052527CD0525282D5 DNA repair protein Rad1 (Rad1) CD0525292D6Identity to S. pombe dim1+,helicase protein 1CD05253045
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