Characterization Of Two Novel Serpins From The Rhipicephalus Haemaphysaloides Tick

Object of this study Rhipicephalus Haemaphysaloides, which is one of the vector for babesia,is one of the main hard tick in south of China. When tick attack the host,two important barrier, one is coagulation cascade, and the other is immune response of host. In order to complete their bloodmeal, tick express a wide range of antihaemostatic molecules and immunosuppressive compounds in their saliva and midgut. Isolation and identification of these molecules make much sense to developing anti-tick vaccine, new anticoagulant, and inflammation inhibitor. Serpins(Serine Protease Inhitors) are a structurally related but functionally diverse family of ubiquitous proteins.Some serpins genes have been isolate from Ixodes ricinus, Ixodes scapularis,Haemaphysalis longicornis and Rhipicephalus appendicularis.Recent studies show that the serpins from the hard tick could inhibit blood coagulation, against the host immune and as anti-tick vaccine to protect host. On this study,there are two novel serpins genes were acquired from cDNAs of half full blood female ticks Rhipicephalus Haemaphysaloides based on the two ESTs by RACE(Rapid Amplificaion of cDNAs ends).These two serpins genes contain the signification feature of serpins family by BLAST analysis,one named RHS1(Rhipicephalus Haemaphysaloides Serpin-1),which has 1280bp and ORF(Open Reading Frame) encode 392aa,there is a signal peptide located on the N-extremity, meanwhile the other named RHS2 (Rhipicephalus Haemaphysaloides Serpin-2),which has 1682bp and ORF encode 380aa,no signal peptide on it. The recombinant proteins of rRHS1 and rRHS2 were expressed by pGEX-4T-1 vector in E.coli BL21(DE3) using 1mM IPTG. and fused protein by GST Sepharose. Genetyx software predict both of RHS1and RHS2 express 69kDa protein contain a 26kDa GST tag in E. coli expression system. By RT-PCR, both of RHS1 and RHS2 transcript in salivary and midgut, especially higher abundance gene in midgut. Two predicted recombinant protein-rRHS1 and rRHS2 were obtained from E. coli expression system by GST affinity chromatography.Western blot showed the two fusion protein were recognized by the anti-Rhipicephalus Haemaphysaloides serum of rabbit. Protein extractive from half full blood female ticks Rhipicephalus Haemaphysaloides were recognized by the anti-rRHSl and anti-rRHS2 by Western blotting, Specific band showed that RHS1 encoded protein expressed in midgut,which larger than prediction size, maybe has been modified in tick.RHS2 encode protein expressed in midgut, which in accord with prediction size. On enzyme assay, chymotrypsin from bovine porcine could be inhibited by the double molar concentrations of rRHS1 or rRHS2, beside it, the thrombin from bovine plasma could be inhibited by the double molar concentrations of rRHS1.