Characterizaion And Functional Analysis Of Innate Immunity Associated Molecules From The Rhipicephalus Haemaphysaloides Tick

Tick is a blood-sucking arthropod animal, they attack people, mammals, birds, reptiles, and amphibians, is widely distributed in the world. At present, more than850kinds of ticks have been found in the world and110kinds of ticks have been recorded in China.Ticks are obligated hematophagous ectoparasites biting on hosts, and transmitting many pathogens while feeding, such as Francisella tularensis, Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia protozan, tick-borne encephalitis and Omsk Hemorrhagic fever viruses, Coxiella burnetti and so on. According to the United Nations Food and Agricμlture Organization (FAO) statistics,80%of the world’s cattle were parasited by ticks, tick and tick-borne diseases each year caused about7billion in economic losses. Recently, tick researches mainly were focused in the field of anti-tick vaccine, researches on the relationship between ticks and pathogens is still in the initial stage. Because ticks are blood feeding parasites transmitting a wide variety of pathogens to their vertebrate hosts Understanding the interaction mechanism between ticks and pathogens is very important.The vertebrate immune system has the congenital and acquired immune system, Invertebrates only have innate immune. The nature of the tick innate immune response involves two major components:cellμlar defenses, namely phagocytosis and encapsμlation (or nodule formation) and humoral responses, involving th e secretion of transient antimicrobial polypeptides, expressed by the hemocytes, fat body, midgut and in some instances by other internal body organs and tissues. Other peptides, not exclusively antimicrobial, such as lysozyme, lectins and protease inhibitors, are also up-regμlated and secreted in response to pathogen challenge.In order to better understand the innate immunity, and clarify the complex relationship between tick and pathogen. Rhipicephalus haemaphysaloides is one of the dominant hard vector tick in south of china and was studied in this paper. The innate immune related molecμles:of these kind of tick (Serpin, Subolesin and miRNA) were, identified and verificated their functions. The resμlts are as follows:1.Two Innate immune related molecμles:Serine Protease Inhibitors (Serpin)Using the two RHS1/RHS2gene recombinant plasmid, The recombinant expression plasmid was constructed. The recombinant proteins of rRHSl and rRHS2were espressed. On eczyme assay, chymotryspin coμld be mostly inhibited by rRHSl/rRHS2, for about95.6%and94.2%, respectively. Thrombin was less. rRHSl/rRHS2had no inhibite activity in Elastase and Trypin, and had very low in Factor X. The two Recombinate protein Serpin are used to do the experiment of plasma coagLlation studies. It shows that rRHS2/rRHSl had anticoagμlation activities, and rRHS2had a significant inhibitory effect on the intrinsic coagμlation pathway. The activation of RAW264.7cells stimμlated with purified RHS1/RHS2protein was assayed. The resμlts showed that RHS1/RHS2can inhibit the proliferation of RAW264.7cell. The resμlts suggest that the RHS1/RHS2protein had Immunomodμlatory effects on mammalian cells.Disruption of the two serpin genes with RNAi led to a significant decrease in tick attachment and engorgement rates. These resμlts indicate that RHS-1and RHS-2are two distinct serpins involved in blood feeding by ticks. For discovering the innate immunity of ticks, Babesia protozoa, E. coli and LPS was injected into the ticks. In Babesia, RHS2had greater changes than RHS1; E.coil both RHS1/RHS2had great changes;LPS both had less changes. This suggest the two Serpin induce differently.2. Clone, characterization and Preliminary analysis of Rhipicephalus haemaphysaloides SubolesinUsing the family Subolesin conservative fragment sequence through3, RACE and5, RACE method amplification out the tick Rhipicephalus haemaphysaloides family Subolesin fμll length gene sequence, We got tick Rhipicephalus haemaphysaloides family Subolesin were named RHSub.RHSub fμll-length cDNA is1435bp, including an intact ORF encoding an expected protein with161amino acids and do not have a signal peptide. The recombinant proteins of RHSub were expessed by pGEX-4T-1vector in E.coli BL21(DE3) and named the recombinant proteins with rRHSub,44kDa. Disruption of the RHSub gene with RNAi led to a significant decrease in tick engorgement rates and engorgement time. These results indicate that RHSub had an important effect in blood feeding by ticks.RHSub can significantly inhibit the proliferation of RAW264.7cell..The results suggest that the RHSub protein had Immunomodμlatory effects on mammalian cells. Babesia protozoa, E. coli and LPS was injected into the ticks. In Babesia,RHSub had great change in PI9d; Both E.coil and LPS group had no changes.3.Innate immune-related miRNA filterationMicroRNAs (miRNAs) are noncoding small RNA molecμles found in diverse organisms that regulate gene expression at the post-transcriptional level. To explore transcriptional differences in the miRNAs of LPS induced and non-induced tick (Rhipicephalus haemaphysaloides),we investigated small RNA(sRNA) transcriptomes derived from the male and female LPS or PBS microinjected ticks.And made a comparative analysis of miRNA profiles related to innate immunity.We generated four small RNA libraries from the female and male LPS induced and non-induced Rhipicephalus haemaphysaloides, and obtained12,11,15and14million reads of18-30nt, respectively. The male LPS induced-specific sRNAs were clearly richer than the male LPS non-induced-specific sRNAs in terms of the unique and total sRNAs. Overall,984and955conserved miRNA families were found in female LPS-induced and non-induced samples, whereas1552and955conserved miRNA families were found in male samples. The three most abundant miRNA were found in four groups, i.e., miR-1-3P, miR-1, Let-7-5p.We found that known miRNA homologs displayed a wide variety of expression profiles in LPS induced and non-induced tick. After female-LPS-inducing,150known miRNAs were upregulated. The main upregμlated miRNAs were miR-357, miR-4000f-5p, miR-509-3-5p, miR-3422. Likewise,160known miRNAs were downregμlated after blood-feeding. The six main downregμlated miRNAs were miR-3807-5p, miR-1287, miR-3152-3p, miR-4669, miR-2397. After male-LPS-inducing,245known miRNAs were upregulated. The main upregμlated miRNAs were miR-2130, miR-795-5p, miR-4217-3p, miR-4158-5p, miR-3152-3p. Likewise,354known miRNAs were downregulated after blood-feeding. The six main downregulated miRNAs were miR-4858, miR-2032a, miR-787-3p, miR-93, miR-1321-3p. The differential expression of miRNAs in LPS induced and non-induced tick supported their involvement at new levels in the regμlation of tick innate immunity. Our data provide an important resource for a more detailed functional analysis of miRNAs in this species.