Construction Of SSH CDNA Library Of Half-blood Male Dermacentor Silvarum And Prediction Of Function Of Differentially Expressed Genes

Dermacentor silvarum is an obligate blood-sucking arthropods that exist in north of China,Russia and Mongolia, also is a portable media that spread tick-borne diseases such as tick-borneencephalitis virus, Bayesian Rickettsia, Siberia Rickettsia, and Bruce bacilli. Dermacentorsilvarum can cause serious harm to humans and animals and cause huge economic losses. Byusing chemistry, biology, genetics and immunology controlling Dermacentor silvarum, the firstthree have achieved some success, but due to drug residues, environmental chemical pollution,biology and genetics are not controllable means immunological control method is nowbecoming a hot tick prevention research.Suppression subtractive hybridization is a PCR-based technique for screening differencesin gene. The PCR-based technique, amplification of differentially expressed cDNA(complementary DNA), while suppressing the total cDNA amplification. Based on thesecondary hybridization kinetics, when homologous hybridizating, single-stranded cDNA athigh abundance annealing faster than the low abundance of cDNA, Isolate the differencesgenes.To construct the suppression subtractive hybridization (SSH) cDNA library of half-bloodmale Dermacentor silvarum and serene the differentially expressed gene in the partial engorgedcondition. The cDNA library of partial engorged half-blood Dermacentor silvarum male wasconstructed in the vector of pMD-18T by the way of SSH. Identified the positive simples bynested PCR, selected8random positive simples, tested weather express in Tester. CalculatedLibrary titers and Recombination rate, screen positive samples sequences by reverse Northernblot.Achieved115positive clones sequenced bacteria samples were sent to sequenced.According to the GenBank database sequence comparison of the sequencing resμLts andbioinformatics analysis, except5repeats, achive12ESTs with partial homology, the remainingEST with low homology in Genbank, and similar homologous fragments are100bp below, thatmeans they are maybe new ESTs. By specμLate homologous functional protein, the differentialexpressed genes are basically the suck blood, blood digestion and so on, including the highexpression of different enzymes, have relation with coagμLation, energy metabolism,transcription regμLation. Meanwhile, part of the ESTs have partial protein homology with theCμLex quinquefasciatus, Drosophila, Ixodes scapμLaris and Rhipicephalus microplus. A low homologous EST1-68（named on the sequences number）was analysised byDNAStar software, based on the5’ and3’,designed two primers for amplification, sequenced byPCR products, spliced the full-length sequence, the ORF contained753bp,encoded250aminoacids, predicted molecular weight of27.61Kda, so renamed DS27. Bioinformatics analysisedthis gene by DNAStar and online software, the gene has a transmembrane region, a signalpeptide,belonging to secreted protein that binding sites of protein kinase, indicating that DS27is closely related to signal transduction. Sequence1825’ deleted, by RACE amplificated825bpof the gene encoding275amino acids with a predicted molecμLar30.7Kda, named DS30.Bioinformatic analysis, the gene without the signal peptide,but transmembrane region,comprising a plurality of protease binding site. Related with energy metabolism and signaltransduction.This research constructed cDNA library of half-blood male Dermacentor silvarum andobtained a new full-length gene successfully, confirmed the success of lirary and reliability,while providing some references for the research of anti-tick vaccine.