Identification Of Cystatins From The Tick Rhipicephalus Haemaphysaloides

Ticks rank first as arthropod vectors of fungi, protozoa,rickettsiae, bacteria, and viruses, causing diseases in nonhuman vertebrates, and rank second only to mosquitoes as vectors of pathogens to humans[14] Ticks and tick-borne diseases affect animal and human health worldwide and are the cause of significant economic losses for animal industry. Ticks function molecular research have very important meaning that at the molecular level to understand the biological characteristics of ticks.Enzyme and enzyme inhibitor molecules plays a very important role in ticks physiology,cystatins are tight-binding inhibitors of papain-like cysteine proteases that cause concern of the majority of scholars.Cystatins are classified, according to certain sequence motifs and the number of conserved cystatin domains, into four subfamilies:the type1cystatins (also known as stefins), the type2cystatins, the type3cystatins (kininogens), and the type4cystatin-like proteins (fetuins, histidine-rich proteins)[50].Family1cystatin is a cytoplasmic protein do not have signal peptide,however the family2is a secretion-type proteins consisting of a signal peptide.In foreign,the cystatins of ticks Haemaphysalis longicornis, Ixodes scapularis and Ornithodoros moubata have been intensively studied, the physiological function of these proteins has been proposed to be the regulation of hemoglobin digestion and tick innate immunity,blood-feeding,as well as immunosuppressive properties.However the domestic to tick cystatins research is blank.The hard tick Rhipicephalus haemaphysaloides were studied,which comes from HuBei province of China and subculture in our laboratory.Through the cystatins relatively conservative in evolution,use of tick Boophilus microplus family1cystatin and Dermacentor variabilis family2cystatin respectively design cystatin conservative area primer and3’RACE primer, obtained the tick Rhipicephalus haemaphysaloides family1conservative fragment and family2cystatin3’end fragment.Using the family1cystatin conservative fragment sequence through3’RACE and5’RACE method amplification out the tick Rhipicephalus haemaphysaloides family1cystatin full length gene sequence, using the family2cystatin3’end sequence through the5’RACE method amplification out the tick Rhipicephalus haemaphysaloides family2cystatin full length gene sequence.Be got tick Rhipicephalus haemaphysaloides family1and family2cystatin were named RHcyst-1and RHcyst-2.RHcyst-1full-length cDNA is471bp, including an intact ORF encoding an expected protein with98amino acids and do not have a signal peptide.Similar with the known family1cystatin, RHcyst-1possesses the N-terminal glycine and the QXVXG segmentPrediction RHcyst-1protein molecular weight is about11kDa, isoelectric point is5.66.RHcyst-2full-length cDNA is773bp, including an intact ORF encoding an expected protein with139amino acids and consisting of a23amino acids signal peptide.Similar to all family2cystatins,RHcyst-2possesses the conserved N-terminal glycine, QXVXG and PW conserved segment,and two disulfide bridges.Prediction RHcyst-2protein molecular weight is about15kDa, isoelectric point is5.20.The recombinant proteins of RHcyst-1and RHcyst-2were expessed by pGEX-4T-1vector in E.coli BL21(DE3) using1mM IPTG.Purification the recombinant proteins by GST resin,and named the recombinant proteins with rRHcyst-1and rRHcyst-2. Using rRHcyst-1and rRHcyst-2to six cysteine protease for enzyme activity inhibition experiment in vitro.The results show that:rRHcyst-1and rRHcyst-2can effective inhibition cathepsin L, B, C, H, S and papain enzyme activity.This study success cloned two new cystatins from tick Rhipicephalus haemaphysaloides, and the inhibition activity was analyzed in biochemical level, and it is have very important meaning for we further understand the physiological function of cystatins in tick Rhipicephalus haemaphysaloides.