Cloning And Prokaryotic Expression Of Serine Protease Gene SP30and SP48from Rhipicephalus Haemaphysaloides

Ticks are ectoparasites that live solely on host’s blood. Ticks are also known fortransmitting a large number of pathogens, including rickettsiae，virus, bacteria andprotozoan, causing diseases in animals and humans. The strategies applicated tocontrol ticks still rely on the chemical pesticides, which causing pesticidal resistanceof the ticks and also environmental problems by the chemical residues. Therefore, it isdesired a new green alternative means to tick control. Unlike other haematophagousarthropods that engorge rapidly, ticks ingest a great deal of host blood during asubstantially long period of attachment, thus they are equipped with an efifcientblood-digestion and nutrient-utilization system. These molecules interrelated to bloodsucking and blood-digestion that may become a new and effective vaccine candidates.But the information are limited on the biochemical pathways interrelated toblood-digestion in the ticks. Serine proteinases were found in a wide range oforganisms and involved in different physiological processes, such as physiologicaldigestion, blood clotting, immune response and apoptosis. A series of enzymesinvolved in the digestion of blood atfer ticks suck a lot of blood. Therefore, theisolation and identiifcation of the enzyme molecules either to understand thephysiological characteristics of the tick or finding some candidates target for againsttick.In this study, we cloned two full-length sequences of the new serine proteasesgenes from the cDNA of partially fed Rhipicephalus Haemaphysaloides by RACE(Rapid Amplification of cDNA Ends). They were named as SP30and SP48. Thebiological analysis and prokaryotic expression of the coding sequence were performed.We analyzed the expression characteristics of the two genes in the different organs and phages using Real-time PCR. Then, RNA interference and Enzyme activityanalysis were performed. The results suggested that SP30cDNA is1194bp and SP48cDNA is1586bp, encoding an expected protein with299and462amino acids,respectively. The predicted molecular weight were30kDa and48kDa, respectively.Both of genes contain signal peptides sequence. They were subcloned into thepGEX-4T-l Exoli expression vector, the recombinant proteins with a molecular massof56kDa and74kDa respectively were obtained by IPTG induction. Antisera weregenerated from by immunized mice using purified recombinant proteins, respectively.Western blot analysis indicated that the antiserum could recognized30kDa and48kDa native protein of the tick in the midgut，indicating high immunogenicity of therecombinant proteins. Real-time PCR analysis showed that SP30and SP48geneswere expressed throughout the developmental stages, but SP30gene was expressedmost richly in unfed nymphae stage, while SP48gene was expressed most richly inpartially fed nymphae stage. The two genes were expressed in different tissues andorgans, both SP30and SP48were expressed most richly in the midgut. The enzymeactivity assay showed that SP48recombinant protein reacted with the substrate in5Real-time PCR showed that the silence of the SP30and SP48gene is effective byRNA interference. For biological characteristics，the ability of replete of tickdescreased rapidly*Above all, we cloned SP30and SP48genes by RACE, and prokaryoticexpression of the coding sequence were performed. We analyzed that the expressioncharacteristics of the two genes in the different organs and phages by Real-time PCR.Atfer that, RNA interference analysis were performed. These research contribute tounderstand physiological characteristics of the ticks and study on the against tickvaccine.