Identification Of Genes From Two Haemaphysalis Ticks Eggs And The Studies In The Immunoprotection Efficacy Against Ticks

Many zoonosis such as Forest Encephalitis, Xinjiang Hemorrhagic Fever and Lyme disease caused by pathogens transmitted by the hard ticks, which seriously threat the health of mankind and the development of husbandry. The control of ticks is emergent question needed to be solved. Currently the principal tick control method in China is the application of acaricides. This approach is, however, associated with a number of disadvantages such as chemical pollution of the food chain and the environment as well as the potential for development of resistance against acaricides by ticks.it is necessary to develop new tick control agent. The study cloned and expressed the gene involed in the development and propagation from Haemaphysalis longicornis and H. qinghaiensis ticks by the method of immunoscreening and cDNA end rapid amplification (RACE). To offer the platform for investigating the molecular mechanism of controlling on the physiological behavior of ticks and evaluating the neotype environmentally friendly pest biological agents and furthermore make up the deficiency in the intervention of physiological behavior and the controlling neotype agent. To develop a new channel for controlling ticks. The mostly results as followed:1) Twenty expression sequence tag (EST) were cloned from the cDNA expression library of H. longicornis egg and H. qinghaiensis by the method of immunoscreening and RACE. Among them, the complete sequence of 12 genes were obtained by the method of cDNA end rapid amplification, which offered the base for investigating the molecular mechanism of physiological behavior of ticks and the screening of the vaccine candidated antigen.2) A fragment of ribosomal protein L24 was obtained from the complementary deoxyribonucleic acid (cDNA) library of H. longicornis eggs. The complete sequence of the clone was subsequently obtained using rapid amplification of the cDNA ends (RACE). Ribosomal protein L24 from H. longicornis had a high percentage similarity to this protein from different species. Conserved domains were also identified in RpL24. Real-time polymerase chain reaction (PCR) analysis showed that this gene is expressed in various tissues and different developmental stages of H. longicornis. Furthermore, HLL24 is mostly expressed in ovaries and salivary glands compared with other tissues in partially fed adult female ticks, and the expression level of HLL24 is significantly lower in eggs and larvae than in other developmental stages. RpL24 was also cloned from H. qinghaiensis and Hyalomma anatolicum anatolicum ticks, respectively. Comparison of their amino acid sequences revealed difference only in several amino acids. A vaccine based on the HLL24 recombinant protein could not protect rabbits against H. longicornis.3) Heat shock protein 70 (Hsp70) was identified from cDNA library of H. longicornis eggs. The Hsp70 cDNA contains 2311 bp that encode 661 amino acid residues with the predicted molecular weight of 72.5 kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmatic reticulum retention signal "KDEL" that is common among ER-localized proteins. The Hsp70 exhibits 90% identity to the putative Hsp70 of Ixodes scapularis,85% to Gallus gallus 78 kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were siginificantly higher than other tested tissues in partially fed adult female ticks. Although the expression level of the Hsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. However, the expression was positively coordinated to the temperature (4℃-37℃, tested). Western Blots analysis showed that the rabbit antiserum against the recombinant H. longicornis Hsp70 protein recognized an about 100 kDa, an about 72.5 kDa, an about 28.4 kDa native protein in egg lysates and 72.5 kDa band was also detected in the protein extracts of partially fed larvae. Immunization using the Hsp70 recombinant protein did not result in the statistically significant reduction of female engorgement and oviposition. These results suggested that although HLHsp70 maybe played a certain role in the physiological activities of ticks, the HLHsp70 as a constitutive protein was not suitable to be selected as vaccine candidate antigen against ticks.4) H. qinghaiensis rabGAP-domain containing protein (TBC1D13) was cloned using rapid amplification of the cDNA ends (RACE). The TBC1D13 cDNA contains 1702 bp that encode 396 amino acid residues with a predicted molecular weight of 46.09 kDa and an isoelectronic point (pI) of 6.0. Phylogenetic analysis of the TBC1D13 from H. qinghaiensis compared with other members of TBC1 domian family 13 members available in GenBank showed that TBC1D13s were evolutionaily conserved and were greatly distant between vertebrate and invertebrate. Real time RT-PCR analysis showed that the expression level of the HqTBC1D13 was significant higher in salivary glands of partially fed females than in other tissues. Western Blot analysis indicated that the rabbit antiserum against the rTBC1D13 recognized an about 49 kDa native protein in salivary glands of partially fed females. Rabbit model vaccinated by rTBC1D13 produced higher antibody titer. Immunization using the recombinant TBC1D13 protein based on the rabbit model resulted in the statistically significant reduction of female engorgement and oviposition. However, it also maybe hed the adverse effect on the rabbit model compared with the control group. So whether the rHqTBC1D13 can be selected as vaccine against ticks is worthwhile to be further evaluated in the other animal model and rabbit model, although the HqTBC1D13 appeared to be important for the survival of ticks.