【作者】 俞英昉；

【导师】 周金林；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2013， 博士

【摘要】 蜱是一种吸血性节肢动物，它们侵袭人、哺乳动物、鸟类、爬行类，甚至两栖类，广泛分布于世界各地。蜱的危害不仅是作为动物常见的外寄生虫，更重要的是作为许多人和动物重要疾病的传播媒介。例如，蜱传动物血液原虫病一直是困扰畜牧业发展的重大疾病,据联合国粮农组织（FAO）统计，世界上有80%的牛被蜱侵袭，蜱及蜱传疾病每年可造成巨大的经济损失。近年来，经蜱传播的新出现和再发传染疾病,如人莱姆病、Q热、出血热和森林脑炎等疾病直接危害着人类健康。国内外对蜱的研究，目前主要集中在抗蜱疫苗研究等领域，相对而言，蜱与病原体之间关系的研究还处于起始阶段。由于蜱的重要性在于它的媒介特征，病原微生物在蜱体内的侵入、移行和发育是一个复杂的生物学过程，了解蜱与病原微生物间相互作用的机制具有重要意义。动物抵御微生物的感染是通过体内一系列细胞和体液免疫反应来实现。脊椎动物具有先天性免疫体系和获得性免疫体系，而无脊椎动物只有先天性非特异免疫反应。目前研究发现，蜱的先天免疫系统十分强大，拥有复杂的细胞免疫和体液免疫，抵抗微生物感染。当有微生物攻击时，除了物理屏障外，蜱还能表达各种抗菌蛋白质、蛋白酶、蛋白酶抑制剂、调理素、抗氧化剂和其他致命的分子,共同构成对病原体入侵的应答。对蜱的抗菌肽、溶菌酶等先天性免疫反应分子的鉴定和功能研究，已取得不少进展。为了更深入了解蜱的先天性免疫反应机制，阐明蜱与病原之间复杂关系，本研究以我国南方的优势蜱种镰形扇头蜱（Rhipicephalushaemaphysaloides）为对象，开展了蜱体先天性免疫相关的蛋白酶抑制分子（丝氨酸蛋白酶抑制分子，Serpin）、调理蛋白（Subolesin）和miRNA分子的分离鉴定和部分功能研究，获得如下研究结果：1.镰形扇头蜱二个丝氨酸蛋白酶抑制分子（Serpin）是具有多重功能的先天性免疫相关分子。利用本实验室已克隆的镰形扇头蜱的二个Serpin分子RHS1/RHS2基因的重组质粒，构建重组表达质粒，完成重组蛋白的表达和纯化，获得重组RHS1（rRHS1）和RHS2（rRHS2）。测定rRHS1和rRHS2对五种丝氨酸酶的抑制活性发现，rRHS1/rRHS2都对胰凝乳蛋白酶（Chymotrypsin）显示最强的抑制活性，分别最大抑制了95.6%和94.2%的酶活性，凝血酶（Thrombin）次之，rRHS1最大抑制65.5%的酶活性，但rRHS2可抑制约30%的凝血酶活性。rRHS1/rRHS2对弹性蛋白酶和胰蛋白酶都没有表现出抑制活性，而对十因子（FactorX）仅有较低的抑制活性。应用APTT和PT试验，进行了二个Serpin分子的抗凝血作用研究，结果rRHS1对内源性凝血途径有显著的抑制作用，而rRHS2没有抑制作用。用重组表达的rRHS1/rRHS2蛋白与巨噬细胞RAW26.7共同培养，发现在1-15μg/ml各个浓度梯度RHS1/RHS2蛋白都有抑制细胞增殖活性，且RHS2蛋白对巨噬细胞有更好的抑制作用，表明RHS1/RHS2对哺乳动物细胞具有免疫调节作用。RHS1/RHS2的RNAi研究发现，与荧光素酶（luciferase）对照组相比，RHS1的24小时吸附率和饱血率分别是42.5%和23.34%，，RHS2是35%和11.67%，对照luciferase是71.67%和35.8%，表明RHS1/RHS2在蜱的吸血过程中有重要作用。但在动物免疫保护和抗蜱疫苗实验，实验组与对照组之间没有显著的变化。通过显微注射方法，将田鼠巴贝西原虫、大肠杆菌和LPS（脂多糖）注射进入蜱体，观察不同病原或免疫刺激抗原诱导的蜱Serpin分子的表达变化，探索其参与先天性免疫反应的特点。结果显示，巴贝西原虫组，RHS1只有在注射后5d较对照组有明显的上升，而RHS2在3，5，9d都有明显上升。大肠杆菌组，对蜱体内RHS1在3，7，9d表达量有显著上升,RHS2在3，5，7，9d表达量明显上升。；LPS诱导后只有RHS1在5d与对照组相比有显著升高。这些结果说明，不同病原对二个serpin分子的诱导反应各不相同。2.镰形扇头蜱Subolsin克隆鉴定和初步分析根据已报道的其他蜱种的Subolesin基因的保守区设计引物，扩增镰形扇头蜱中的Subolesin的保守片段。利用得到的Subolesin保守片段序列，通过3’RACE序列和5’RACE方法克隆到镰形扇头蜱Subolesin全长基因序列，命名为RHSub。RHSub的全长基因为1435bp,具有一个编码161个氨基酸的开放阅读框，没有信号肽序列，推测RHSub蛋白的大小为44kDa，将RHSub的ORF亚克隆至PGEX-4T-1表达载体，转化BL21感受态细胞，成功进行重组表达。RNA干扰实验发现，实验组与对照组相比饱血率和饱血时间都有显著的下降，表明RHSub在蜱的吸血过程中有重要的作用。用重组表达的Subolesin蛋白与巨噬细胞RAW264.7共同培养，在15ug/ml,7.5ug/ml浓度有显著的抑制活性。结果提示Subolesin蛋白对巨噬细胞很好的抑制作用。蜱注射病原实验显示，注射Babesia原虫，攻毒后第9天Subolesin的表达量有显著的上升，表明与巴贝斯虫感染的先天性免疫直接相关。3.先天性免疫相关的miRNA分子筛选脂多糖LPS是脂质和多糖的复合物，为革兰氏阴性细菌细胞壁的主要成分，LPS能激活节肢动物的先天性免疫途径。本研究通过显微注射方法，分别向镰形扇头蜱雌蜱和雄蜱中注射PBS和LPS，通过检测蜱中的小RNA（sRNA）转录组，比较分析LPS诱导后与先天性免疫有关的miRNA。结果表明：（1）.构建了4个小RNA文库，包括雌蜱LPS注射组，雌蜱PBS注射组以及雄蜱LPS注射组和雄蜱PBS注射组，分别得到1.2千万，1.1千万，1.5千万和1.4千万个18-30nt的基因数据。（2）雌蜱LPS注射组，雌蜱PBS注射组以及雄蜱LPS注射组和雄蜱PBS注射组分别有984，955，1552，955个保守的miRNA家族分子，其中各组最大丰度的3个都是miR-1-3P,miR-1,Let-7-5.(4)。（3）与对照PBS组比较，雌蜱组LPS诱导后有150个miRNA上调，主要有miR-357,miR-4000f-5p,miR-509-3-5p,miR-3422；有160个下调，主要有miR-3807-5p,miR-1287,miR-3152-3p,miR-4669,miR-2397。与对照PBS相比，雄蜱组LPS诱导后有245个miRNA上调，主要有miR-2130,miR-795-5p,miR-4217-3p,miR-4158-5p,miR-3152-3p；有354个下调，主要有miR-4858,miR-2032a,miR-787-3p,miR-93,miR-1321-3p。这些LPS注射后差异表达miRNA作为蜱先天性免疫相关候选分子，可能调控蜱的先天性免疫过程，为进一步研究镰形扇头蜱的先天性免疫miRNA分子提供基础。更多还原

【Abstract】 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.更多还原