Recombinant Expression Of The Anticoagulant Molecules From Tick Rhipicephalus Haemaphysaloides In Insect Cell And Its RNA Interference

Ticks are obligate haematophagous ecto-parasites that solely live on host blood throughout their live .They are known to ingest a large blood-meal during a prolonged period of feeding. Ticks are considered as economically and hugienically important pests for their direct harmful effects to the host, such as damage to the skin, causing anemia and loss of vigor, and also for their vector competence in transmitting various kinds of pathogens of humans and animals. In order to get blood duratively, they secrete some biological proteins into hosts'blood, such as inhibitors of blood coagulation, inhibitors of immunity, adhensive proteins, toxins, antibacterial proteins. These proteins play a key roll in the process of tick hemophagia and transmitting other pathogens.Our group do research on the inhibitors of blood coagulation from tick to find novel strategy for protecting tick and tick-brone diseasesSelect two EST sequences named Rhipilin-1 and Rhipilin-2 from salivary gland subsracted cDNA library of Rhipicephalus haemaphysaloides half-fed female constructed by Vector and vector-borne disease group. Then the two genes are cloned into the plasmid of pFastbac HTA to eukaryotic expression and identification the protein by indirect immunofluorescence and Western blotting . We got a 24.5kDa recombinate protein Rhipilin-1 and a 28.3kDa recombinate protein Rhipilin-2. After purified and dialysed , recombinate protein Rhipilin-2 are used to do the experiment of plasma coagulation studies and enzyme amidolytic activity inhibition assays. It shows that recombinate protein Rhipilin-2 which can increase the APTT time of rabbit plasm have obviously anticoagulation activities, meanwhile recombinate protein Rhipilin-2 has strongly inhibitions on serin protein and concentration dependent, such as human elastase and trypsin ,but less inhibitory effects on bovine thrombin.Besides , we tried to knock down Rhipilin-1 and Rhipilin-2 gene in adult ticks by immersed in dsRNA and observed fed weight , the rate of adhesion , the number of eggs , hatchability . When we knock down the the two genes , the ability of adhesion and replete time of tick descreased rapidly , as well as the number of eggs . So,the two genes have a key roles in the process of tick sucking blood.Above all , research in this article involved eukaryotic expression(baculovirus express system) of Rhipilin-1 and Rhipilin-2, and make a further study of Rhipilin-2 fusion protein, meanwhile we analysed the effect of knocking down Rhipilin-1 and Rhipilin-2 genes. All of this has made a substantial foundation to the further study .