| 其他摘要 | Ticks are blood-feeding ectoparasites of vertebrates. When feeding on vertebrate host, ticks induce both innate and specific acquired host-immune responses as part of anti-tick defenses. Host immune defense against ticks can reduce tick viability, sometimes resulting in tick death. Tick responds to the host immune attack by secreting saliva containing pharmacologically active molecules and modulating host immune response. Tick saliva-effected immunomodulation at the attachment site facilitates both tick feeding and enhances the success of transmission of pathogens from tick into the host. On the other hand, host immunization with antigens from tick saliva can induce anti-tick resistance and is seen to be able to induce immunity against pathogens transmitted by ticks. Hyalomma asiaticum asiaticumis a main kind of hard ticks in China, which is widely distributed in Xinjiang, Neimenggu and Ningxia. They are vectors of many protozoan and pathogens, such as Theileria annulata andCrimean-Congo hemorrhagic fever virus. In this study, two immunoregulatory peptides, hyalomin A1 and -B1 were identified from salivary glands of hard tick Hyalomma asiaticum asiaticum by gel filtration and RP-HPLC. Edman degradation method was used for the determination of their amino acid sequences. The determined sequences of hyalomin A1 and -B1 were QTPRTIGPPYT and TLRTTTDYSTTVENGNLTTPAANSTEKGNGLYGL, composed of 11 and 34 amino acid residues, respectively. They are small peptides with molecular weights of only 1231.16 and 3688.2 by MALDI-TOF-MS analysis. They are not found to be similar to any proteins already known. The cDNA cloning result confirmed that these two peptides are encoded by a specific gene, which is composed of 801 bp. This cDNA encodes a precursor protein composed of 221 amino acid residues. In the sequences of the 221-amino acid precursor, there are three copies of hyalomin A1 and three homologs of hyalomin B1. Hyalomin A1 is composed of 11 amino acid residues, hyalomin B1, B2 and B3 is composed of 34, 33 and 32 residues, respectively. All of the sequences of mature hyalomin A and B peptides are flanked by the possible enzymatic processing site of -RR-. The possible immunomodulatory activities of hyalomin A1 and -B1 were studied. Both hyalomin A1 and -B1 can exert significant antiinflammatory functions, either by directly inhibiting host secretion of inflammatory factors such as tumor necrosis factor-α, monocyte chemotectic protein-1, and interferon-γ or by indirectly increasing the secretion of immunosuppressant cytokine of interleukin-10. Hyalomin A1 and -B1 were both found to potently scavenge ABTS+ free radical in vitro in a rapid manner. The antioxidant activities of hyalomin A1/B1 may contribute to their immunoregulatory and anti-inflammatory abilities. The effects of hyalomin A1 and -B1 on the LPS-stimulated phosphorylations of ERK1/2, SAPK/JNK, p38 MAPK kinases and degradation of IκB-α in RAW 264.7 macrophage cells were examined using western immunoblot analysis. The JNK/SAPK subgroup of the MAPK signaling pathway was involved in such immunoregulatory functions of hyalomin A1 and -B1. Given the inhibitory abilities of hyalomin A1/B1 on MAPK activation, we tested their effects on mouse splenocyte proliferation, hyalomin A1 could slightly inhibit splenocyte proliferation and hyalomin B1 showed cell proliferation inhibitory effects at low concentrations. Cell viability has not been affected by hyalomin A1/B1, indicating that the immunoregulatory properties of hyalomin A1/B1 are not related with cytotoxicity. Freund’s complete adjuvant induced inflammation model was used to evaluate the potential antiinflammatory ability of hyalomin A1 and -B1, the results showed they inhibited adjuvant-induced inflammation. Moreover, both hyalomin A1 and -B1 had decraesed effects on LPS-induced TNF-α and IL-6 mRNA expression. These results showed that immunoregulatory peptides derived from tick salivary glands suppress host response by modulating cytokine secretion and scavenging reactive oxygen species, indicating these peptides could help the ticks suppress host inflammatory responses and successfully get a blood meal. |
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