Study On Differentially Expressed Genes In The Salivary Gland Of Hyalomma Asiaticum Tick

60 Hyalomma asiaticum female adult ticks selected form a laboratory colony are divided into two groups. The salivary glands of the unfed and partially engorged ticks are dissected. After the the total RNA preparation, the first strand cDNA synthesis and its amplification by LD-PCR, the Rsa l-digestion of the cDNA, the ligation of adapter-1 or adapter-2R to tester cDNAs, subtractive hybridizations and selective amplifications, the differentially expressed sequences have been Isolated. The gel-purified fragments from the secondary PCR amplification are lagated into pGEM T-Easy vector, and the resulting recombinant Plasmids are transformed into DH5acompetent cells. Screening and sequencing present 120 valid sequences, 84 of them belong in expressed sequence tags (ESTs). The RT-PCR result confirms that 10 fragments randomly selected from the library are expressed in the feeding female tick's salivary glands, but are not in the salivary glands of unfed female ticks.Sequences from the library are analyzed for homology in NCBI databases, which reveals that 90 of the 120 sequences in the library have significant matches to some other genes in databases, 21 of those being similar to the genes from other ticks, 19 showing due homology with the genes from other parasites. Seven of the 21 cDNA being similar to tick genes, 2 with ACGCGGGG and 5 with Poly(A), are ESTs; and two of them are full-length cDNAs, and one is regarded as new gene (DQ021902) . HaB1 and Ha7.7 are two ESTs selected from the 7 ESTs above.HaB1-GSP₁ and HaB1-GSP₂, two primers for the unknown end of HaB1, are designed on HaB1 sequence. HaB1-GSP₁ and UAP are used to amplify the first strand cDNAs produced by reverse transcription of the total RNA of H. asiaticum female tick's salivary, the products are re-amplified with HaB1-GSP₂ and AUAP, then the 3'-end partial cDNA of HaB1 at has been obtained. Based on the sequence combined HaB1 with its 3' -end, a pair of full-length primers for GP₂₉, HaB1-FLP⁺ and HaB1-FLP^- are created. a new gene(GP₂₉, AY803896) is subcloned by amplifications of the first strand cDNAs. As described above, three primers for the unknown end of Ha7.7 (Ha7.7-GSP₁, Ha7.7-GSP₂, Ha7.7-GSP₃) and a pair of full-length primers for GP₁₅ (Ha7.7-FLP⁺ and Ha7.7-FLP^-), are designed.the 5'-end partial cDNA of Ha7.7 and GP15 (DQ020265) has been generated.The open reading frame of GP29 is cloned into expression vector pGEX-4T-1 in order toconstruct the recombinant plasmid. After The transfectant cells DH5a or BL21 being induced, A novel protein, corresponding to the expected in mass, is expressed?The peptide mass mapping and the peptide sequencing suggest that there are 8 peptide fragments from GST and 2 peptide fragments matching the deduced of P29 in the tested sample? SFDILQFLQDR is the sequence of the peptide with 1381.5228m/z. To sum up, the protein in the 53000 band of SDS-PAGE is a fusion protein comprised of GST and GP29.the open reading frame of GP15 ligate the linear expression vector pET32a(+), then the recombinant plasmid is yielded = A novel protein about 36000 molecular mass is expressed by the Escherichia coli (BL21 strain )o This protein could be purified by HisBind resin, and could be recognized by the antisera against the fusion protein. The expression type analysis shows that a fusion protein is expressed in Escherichia coli as inclusion bodies.