The Influence Of Temperature Change On Cysteine Protease Activity Of Three Kinds Of Hard Ticks

Hard tick is a class of ectoparasites that can parasitize in a variety of warm-blooded animals (including humans). The hosts’ blood is the only source of nutrition. As the characters of sucking more and biting a long time and repeatedly, the livestock which were bit will manifests as symptoms of coat scattered, skin ulcers, necrosis, emotion agitation and loss of appetite. In addition, ticks can also spread a variety of pathogens such as bacteria, virus, protozoa and reckettsia resulting the zoonotic disease.The life cycle of hard tick is complex, so domestic and foreign scholars committed to the study of biological characteristics of hard tick. At present, global warming makes a significant effect in ticks and tick-borne diseases. High temperature not only expands distribution of the tick, but also promotes the prevalence of tick-borne diseases. Many scholars at home and abroad have done large number of studies in tick classification and biological characteristics, but the molecular mechanisms of the regulatory of the temperature to biological characteristics of tick has not clear yet. Cysteine protease is recognized as a new anti-tick antigen candidate molecule all over the world, also an important metabolic enzyme, thus the present study focus on the relationships between the cysteine proteases of three kinds of hard ticks and temperature.In this study, conservative fragments of cysteine protease of three kinds of hard ticks were cloned and homology analyzed firstly. Secondly, the CP of R.microplus was expressed prokaryotic and the in vitro activity was then analyzed. Finally, the effect on activity of three kinds of CP in vivo in different temperatures was analyzed after identifying the appropriate keep temperature in laboratory.1) Obtainment of the conserved sequence of cysteine protease of three kinds of hard ticksUse specific primer to amplify, clone the conserved sequence of cysteine protease from the cDNA of the R.haemaphysaloides, H.asiaticum kozlovi and R.microplus. The sequence result showed that the amplified fragments from the three kinds of hard ticks contain a conserved area, and the gene sequences has more than80%homology with the sequences of cysteine protease of Rhipicephalus and Dermacentor and70%homology with the sequences of cysteine protease of Haemaphysalis in the NCBI.2) Cloning, expression and activity analysis of the R.microplus cysteine proteaseThe primers of the R.microplus cysteine protease were designed, its ORF was cloned, pET-28a-cys prokaryotic expression vector was build and then pET-28a-cys was expressed in BL21. The activity of recombinant protein was detected after self-activation. The results showed that full length sequence of the cysteine protease is1041bp which encoding332amino acids, and it is98%homology with the R.microplus cysteine protease gene in NCBI. The proteolytic activity of the recombinant protein was only stimulated after self-activation under the acidic condition.3) Identification of the optimum growth temperature of hard ticks’Under laboratory conditions, R.haemaphysaloides and H.asiaticum kozlovi was fed at22℃,28℃,37℃respectively. The differences of biological characteristics among each tick group were observed. The results showed that the ecdysis rate of both the two kinds of larva and nymph and the hatchability of two kinds of eggs were best in28℃.4) The effect of different temperatures on the activity of three kinds of hard ticks cysteine proteaseThree temperatures (24℃,28℃,32℃) were chose to keeping the three kinds of hard ticks. Cysteine protease in vivo was extracted after keeping Id,3d,5d, and the proteolytic activity of each group were compared. The results demonstrated the relationship between the temperature and the activity of cysteine protease was existed. In the range of24℃to32℃, in the same keeping time, the activity of cysteine protease from larva and nmyph gradually decreased as the temperature rose. Therefore, the increasing temperature promotes the metabolism of CP within hard ticks, which is accelerated the utilization of CP in vivo.The results in the present study give an important rationale of prevention and control of ticks and tick-Borne disease under the trend of global warming. Cysteine protease may become a new drug target for prevention and treatment of tick-borne diseases.