| Arthropoda is the largest phyla in the animal kingdom.There are more than 120 millions species of arthropod accounts for more than 80%world's existing animals.Many arthropods use their unique venom or saliva system for hunting.In this thesis,two bioactive peptides from N.clavata spider and I.scapularis tick were studied based on their survival strategies.The spider N.clavata belongs to genus Nephilinae,family Tetragnathidae,order Metathelae and the class Insecta.N.clavata is widely distributed all around the world,especially many provinces of China.The spider N.clavata makes huge web in the bushes,orchards and gardens for preying insects,and is known no obvious toxicity to mammal.Previous study about the venom of the spider N.clavata focused on the compounds of the polyamines,and there have been no reports of the peptidic components of the venom.Bio-pesticides from natural organisms are promising alternatives to chemical insecticides based on their specificity.As an important source of biopesticides,the development of spider venom insecticide polypeptide resources can expand the bio-insecticide resource library.Ticks are the second largest disease vector besides mosquitoes and they can be short parasitic on the vertebrate body surface.In the process of feeding,ticks can transmit a variety of pathogens.The I scapularis ticks are feeding longer than other blood sucking arthropods and need to be fully saturated on the surface of the host for several days.Its saliva contains a variety of active molecules which can fight the coagulation and immune response of the host.The active proteins in ticks' saliva are usually induced during feeding and enhance the success of transmission of pathogens from ticks into the host.Borrelia burgdorferi sensu lato(also known as Lyme spirochetes)is the most famous pathogen of Lyme disease.B.burgdorferi has a unique structure and has the strongest mobility so far found in the microbial kingdom.Due to the high variability of tick-borne B.burgdorferi antigens,the vaccines of tick-borne infectious diseases have not been developed yet.In order to understand the molecular mechanism of pathogen-tick-host and to clarify the molecular mechanism of tick-borne pathogens from vector to host,it will provide an important theoretical basis for the prevention and control of tick-borne infectious diseases.The aim of the study is to further elucidate the mechanism of their venom systems in the predation process and to screen for active peptide precursor molecules with application potency.In the experiment part of this thesis,the peptidic components in the venom of the spider N.clavata were isolated and purified.The insecticidal activity of the venom and the components were tested with cockroaches.Fraction I,which had higher insecticidal activity,was further isolated.By tracking the insecticidal activity of each peak,a peptide toxin?-NPTX-Nc1a with strong insecticidal activity was identified with a half-lethal dose of 135 pmol/g to Periplaneta americana.After the partial sequence of Nc1a was obtained by edman degradation,we designed primers to amplify the cDNA library of the spider venom gland.The whole sequence of Nc1a was obtained by sequencing and the molecular weight and sequence of the peptide was further identified by mass spectrometry and secondary mass spectrometry.We observed rapid paralysis and lethality when injected spider venom into cockroaches,suggesting that the venom might act on the nervous system of insects.We isolated the DUM neurons of P.americana and found that Nc1a could inhibit the sodium current by 57%± 5.5%and the potassium current by 24.6%±6.8%which could lead to paralysis and death of insects quickly.At the same time,Nc1a and the spider venom had no effect on the voltage-gated ion channels of rat DRG neurons and human acid-sensitive ion channels.In conclusion,the peptide Ncla,which is found in the venom of the spider N.clavata,can specifically act on the voltage-gated ion channels of insects and has no effect on mammalian ion channels.It is a safe and potential bio-insecticide molecule.Next,we found a protein Salp19 which is identified from the salivary glands of I.scapularis on the basis of previous study.Firstly,it was confirmed that Salp19 had significant difference before and after the blood feeding by testing the tissues of the ticks at different time points after feed.Next,we performed high-throughout protein microarray analysis in which more than 19000 human protein probes were detected,Salp19 preferentially bound to lymphotoxin receptor.Lymphotoxin(LT)is widely expressed in all celltype surfaces except T lymphocytes and B lymphocytes,and the multimerization of lymphotoxin receptor is necessary for signal transmission.Based on the results,we further confirmed that Salpl9 can bind and inhibit the multimerization of lymphotoxin receptor by many means.In the early stage of B.burgdorferi infection of human PBMC a large number of IL-22 secretions can be induced.We examined the effects of Slap 19 on the infection of B.burgdorferi in vitro by mononuclear cells(PBMC)isolated from human peripheral blood.And we also examined the classical and noncanonical NF-?B signaling pathway activated by spirochetes infection.It was found that Slap 19 can inhibit noncanonical NF-?B signaling pathway and can significantly inhibit the secretion of IL-22.We also examined the effect of Salp19 on the stimulation of THP-1-induced macrophage-induced cytokines by B.burgdorferi.Salp19 have inhibitory effects on IL-1? and GM-CSF secretion.IL-22 and IL-17 are mainly secreted by the activated T cell subtype(Th17)and play an important role in the host's resistance to exogenous pathogens.We choose Balb/c mouse strain and B.burgdorferi B31-A3 wild-type spirochete strain as the experimental subjects.Different doses of Salp19 protein were tested for B.burgdorferi infection in Lyme disease mouse model.Mice were sacrificed and samples of plasma and susceptible tissues were collected.Quantitative PCR and other results showed that there were significant spirochete infections in spleen and joint.The number of spirochete copies was significantly higher than that of BSA control group.We detected the expression of multiple inflammatory factors in mice by high throughput of mouse cytokine chips.The results showed that Salp19 could significantly affect the expression of IL-6,IL-8,CXCL13,GCSF,MIP-1?,CCL5 and other cytokines and chemokines in Lyme disease mice.In order to explore the mechanism of Salp19 promoting B.burgdorferi infection,we investigated the effect of Salp19 on the maturation of splenic DC cells and the differentiation of T cells(Th17)in mouse model by flow cytometry.We found that Salp19 significantly inhibited the maturation of DC cells in mice infected with B.burgdorferi.The results showed that Salp19 also significantly inhibited the differentiation of Th17 cells.In this paper,the function of two bioactive proteins derived from two toxic arthropods was studied based on the survival strategies.Both of them have great potential for application.The peptide toxin Ncla,which had strong insecticidal activity as 135 pmol/g to cockroaches.We found Ncla acts on the insect sodium and potassium channels.Salpl9,a protein that promotes the infection of B.burgdorferi,was identified from the salivary gland of the ticks.Based on the study of the immunosuppressive mechanism of Salp19,it was found that Salpl9 could act on lymphotoxin receptor and inhibit the signal transduction of the receptor.In this paper,two molecules with potential for development and application were identified from two toxic animals and the structure and function of the two proteins were deeply studied.In addition,the methods used in this thesis will be helpful to the development of other toxic animal natural active resources. |
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