| Haemaphysalis longicornis, as the most important obligate ectoparasite, are one of the vectors transmitted bacteria, virus, protozotan, rickettsia, and so on. It can seriously threat the health of mankind and livestock, restricts the development of the livestock industry and cause significant economic losses in China. Though the principal acaricides can provide a degree of protection to tick control, however, this approach is associated with lots of disadvantages such as chemical pollution of the food chain as well as the potential for development of resistance against acaricides of tick, which makes that it is necessary to search for an alternative biological method of tick control. During the recent years, the use of vaccines provide a new approach to biological tick control, while this approach mainly depends on the identification of protective antigens in ticks. This study obtained the full length of unknown gene by rapid amplification of the cDNA ends (RACE), described the cloning and expression, analyzed the distribution and biological function, and finally assessed the potential for the development of vaccine against ticks.The full-length cDNA encoding acid phosphatase (HL-3) from the tick H. longicornis, was obtained by 5′RACE. The full-length of HL-3 was 1324 bp, and contained a 1137 bp open reading frame (ORF) coding for 356 amino acids with a predicted theoretical isoelectric point (pI) of 6.35 and molecular weight of 41.0 kDa. The gene included three N-glycosylation sites, a putative signal peptide, and many phosphorylation sites. Additionally, the 3D model structure of HL-3 based on the crystal structure of human prostatic acid phosphatase was constructed by Swiss-Model. The complete acid phosphatase cDNA sequence from H. longicornis has been submitted to the GenBank database under the accession number HM150759.The recombinant plasmid of pGEX-4T-1/HL-3 was expressed in E. coli. The rHL-3-GST recombinant protein exhibited the strong reactogenicity by Western blotting. The enzyme could hydrolyze para-nitrophenyl phosphate (pNPP) substrate at an optimum pH of 5.0. Real-time PCR analysis showed that the HL-3 transcripts were expressed in various stages of H. longicornis ticks and were significantly induced by blood feeding. Furthermore, the expression of HL-3 in midguts was significantly higher than in other tested tissues of partially fed adult female ticks. In addition, the transcripts of the HL-3 mRNA in LPS-injected adult ticks and Theileria sergenti infected larvae was significantlly expressed. The analysis of immunoblotting showed the native protein of HL-3 was existence in various stages of ticks. Vaccination of rabbit with rHL3 and RNAi all confirmed that the protein could affect the engorgement weight and mortality, and there were no apparent differences in other parameters between the treated and control groups.The complete cDNA encoding misexpression suppressor of kinase suppressor of Ras (KSR) (HL-4) from the H. longicornis, was obtained by 5′and 3′RACE. The full-length cDNA was 1428 bp, and contained a 1185 bp ORF that encodes 395 amino acids with a predicted molecular weight of 44.57 kDa and theoretical isoelectric point (pI) of 6.21. The sequence included five N-glycosylation sites without putative signal peptide and transmembrane domain, and was considered to be an outer membrane protein. The cDNA sequence from H. longicornis was submitted to the GenBank database under the accession number HM195183. The gene was cloned into the pGEX-4T-1 vector and expressed in E. coli. The expression product of HL-4 showed the strong reactogenicity by Western blotting. Real-time PCR analysis showed that the gene was expressed in different developmental stages of H. longicornis, and was significantly induced by blood feeding. Furthermore, the HL-4 transcripts were mostly expressed in ovaries and salivary glands compared with other tissues in partially fed adult ticks. Finally, the transcripts of the HL-4 mRNA in LPS-injected and T. luwenshuni infected ticks was lower than in control groups. This is the first report about the gene associated with KSR in Ras pathway of ticks.
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