亚洲璃眼蜱唾液腺两个免疫调节多肽的分离纯化、基因克隆与功能研究
文献类型：学位论文
| 作者 | 武静 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赖仞 |
| 关键词 | 蜱 唾液腺 多肽 免疫调节 抗氧化 抗炎活性 |
| 其他题名 | Purification, Cloning and Function of Two Immunoregulatory Peptides from Salivary Glands of Tick, Hyalomma Asiaticum Asiaticum |
| 学位专业 | 动物学 |
| 中文摘要 | 蜱是一类吸血的体外寄生虫，当它们吸附在脊椎动物身上吸血的时候会激活宿主的先天和特异性免疫反应。宿主的这种免疫排斥反应将会降低蜱的存活率，甚至导致蜱的死亡。蜱为回应宿主的免疫攻击，其唾液腺将会分泌富含药理活性的分子来调节宿主的免疫反应。蜱在宿主吸血部位分泌的唾液所引起的宿主免疫调节反应，既有利于蜱顺利地进行吸血，又能将其携带的病原体成功地传播至宿主。因此，用来源于蜱唾液的抗原免疫宿主能使宿主对蜱产生排斥，同时也能诱导宿主产生阻碍蜱携带的病原体传播的免疫反应。 亚洲璃眼蜱Hyalomma asiaticum asiaticum是我国的主要蜱种，广泛分布于新疆、内蒙和宁夏地区，携带多种原虫和病原体，主要传播环形泰勒虫和新疆出血热病毒。 在本研究中，我们通过凝胶过滤和反相高压液相层析从亚洲璃眼蜱的唾液腺得到两个具有免疫调节功能的多肽hyalomin A1和-B1。通过Edman降解法测定它们的氨基酸序列分别为QTPRTIGPPY（11个氨基酸残基）和TLRTTTDYSTTVEN GNLTTPAANSTEKGNGLYGL（34个氨基酸残基）。MALDI-TOF-MS分析它们的分子量分别为1231.16和3688.2 Da。经比对发现它们与数据库中的已知蛋白质没有相似性。 cDNA克隆的结果确定多肽hyalomin A1和-B1由一个特异的基因编码，该cDNA序列长度为801 bp，它编码的前体蛋白由221个氨基酸残基组成，其中含有3个相同的hyalomin A1拷贝和3个hyalomin B1类似物的拷贝。Hyalomin A1由11个氨基酸组成，hyalomin B1由34个氨基酸残基组成，hyalomin B2由33个氨基酸残基组成，hyalomin B3由32个氨基酸残基组成。在整个基因编码的前体蛋白序列中，成熟的hyalomin A与hyalomin B均由可能的酶切位点RR（精氨酸－精氨酸）或者RRR（精氨酸－精氨酸－精氨酸）分隔开。 我们对hyalomin A1和-B1可能具有的免疫调节活性进行了研究，发现hyalomin A1和-B1均具有显著的抗炎功能。它们直接抑制促炎细胞因子如肿瘤坏死因子-α，单核趋化蛋白-1和干扰素-γ的分泌，或者间接促进免疫抑制细胞因子白介素-10的分泌。Hyalomin A1和-B1都能快速清除体外ABTS+自由基，我们推测它们的抗氧化活性可能有助于其免疫调节和抗炎能力。Western Blot检测发现hyalomin A1和-B1能抑制LPS诱导小鼠单核巨噬细胞系RAW 264.7产生的ERK1/2、SAPK/JNK、p38 MAPK激酶的磷酸化以及IκB-α的降解。MAPKs信号通路中的JNK/SAPK参与了hyalomin A1和-B1的免疫调节功能。由于hyalomin A1/B1抑制MAPKs的激活，我们检测了它们对小鼠淋巴细胞增殖的影响，发现hyalomin A1能微弱地抑制淋巴细胞增殖，而hyalomin B1在低浓度时抑制淋巴细胞的增殖。Hyalomin A1/B1对细胞活力没有影响，表明它们具有的免疫调节功能与细胞毒性无关。用完全弗氏佐剂诱导的炎症模型来评价hyalomin A1/B1所具有的抗炎能力，结果表明它们能抑制佐剂引起的炎症。此外，还发现hyalomin A1/B1能抑制LPS诱导的TNF-α和IL-6的mRNA表达。这些结果表明从蜱唾液腺来源的免疫调节多肽hyalomin A1/B1能通过调节细胞因子的分泌，清除活性氧自由基来抑制宿主的免疫反应，提示这些多肽可能有助于蜱抑制宿主的炎症反应，从而成功获得血餐。 |
| 英文摘要 | 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. |
| 语种 | 中文 |
| 公开日期 | 2013-04-23 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7385]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 武静. 亚洲璃眼蜱唾液腺两个免疫调节多肽的分离纯化、基因克隆与功能研究[D]. 北京. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：昆明动物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。