【作者】 刘光远；

【导师】 陈怀涛； 才学鹏；

【作者基本信息】 甘肃农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 硬蜱科的蜱能够传播许多种疾病，是吸血媒介中仅次于蚊类（Mosquitoes）排在第二位的极其重要的人类和动物疾病的传播媒介。它们具有地理分布范围广、气候适应能力强以及叮咬宿主的多样性的特点，是一个极为成功的生物种群。硬蜱属蜱的生活史与它们的宿主特异性有关，在长期的进化和寄生生活中形成某些针对其宿主的机能来抑制止血和免疫应答。 Ribeiro记载，蜱具有很强的能力逃避或抑制宿主的凝血系统，使其对宿主的选择性减到最小。另外一些蜱在唾液中需要一些特殊的拮抗物，用来逃避宿主的免疫反应。同时，Wang等研究认为，雄蜱可以帮助雌蜱吸血。刺激宿主对蜱的免疫反应的主要抗原决定簇来源于唾液，由蜱的唾液腺分泌。唾液腺是蜱体内最大的腺体，从一开始吸血，蜱就将唾液注入宿主体内，大多数硬蜱的唾液中包含有粘结混合物，用来将蜱的口器固定于宿主的皮肤，各种各样的酶和不同类型的生物活性分子也来自于蜱的唾液腺和唾液。这些成分是获得成功吸血的基础，它们使血液不断进入叮咬部位，抵抗宿主的止血剂和炎症介质以及帮助蜱逃避宿主的排异反应。唾液腺成分分析表明它们有调节宿主的细胞因子应答和降低淋巴细胞对T-细胞有丝分裂应答的能力，蜱通过唾液分泌的麻醉毒素也能引起宿主的疾病甚至死亡。唾液腺也是蜱传病传播的主要器官。此外，这些活性成分提供的唾液活性传递(SAT)在非毒血症蜱传性病毒病的传递中起重要的作用。蜱的唾液腺蛋白组分的研究已成为生物学、免疫学和生物药学研究和开发的主要目标之一，从蜱的唾液腺中已经发现了许多对蜱的免疫和生物制药有用的蛋白，如组胺结合蛋白(Histamine-Binding protein)、IgG结合蛋白(Immunoglobulin binding protein) 、IL-2结合蛋白(IL-2 Binding protein)、 p29蛋白、HL34和35蛋白等。对有些蛋白的研究已有了突破性进展，已分离、克隆和表达了多种蛋白,某些蛋白已测出了其模拟结构。IgG结合蛋白的研究已得到了许多学者的重视，从各种蜱中分离出了多个蛋白，目前的工作主要集中在已知蛋白的重组表达和功能测定上。Wang等从附尾扇头蜱的唾液腺中分离、测定和表达了三个IgG结合蛋白，并预测了他们的功能。本项研究旨在观察附尾扇头蜱在不同的饲喂方式下，雌、雄蜱的吸血能力，唾液腺分泌蛋白组分的变化情况，雌、雄蜱不同体重配对时各自的吸血情况、雌蜱的卵块重量和个数、饱血天数以及雄蜱不同体重下的唾液腺分泌蛋白的多态性，不同叮咬天数雄蜱唾液腺中IgG结合蛋白的分布情况以及它们的分离和序列测定等内容。利用统计学方法分析各组数据的差异性和规律，推测其生物学意义。经研究获得以下结果：1．雄蜱无论配对与否，配对方式如何，各组相同天数的蜱相比较，吸血程<WP=6>度无显著变化，雄蜱分泌的生物活性物质仅仅存在于叮咬点，当与雌蜱配对时，方可发挥生物活性的联合作用。或许生物活性物质并不能进入宿主的体液循环，经体液传递到达雌蜱的叮咬点起联合作用；抑或是进入体液循环的量很少，不足以引起蜱的联合作用。说明雄蜱帮助雌蜱吸血的功能是有条件的，联合吸血作用是雌、雄蜱在叮咬过程中达到严格意义上的联合方可发挥作用。2．雌蜱的体重增加明显，自第6天起，三个组的雌蜱在体重增加程度上有明显差异，单一雌蜱组和雌、雄同体不混合组雌蜱虽体重增加达3倍以上，但在叮咬第6～10天之间，体重趋于相对恒定状态，始终叮咬于动物体，并不饱血脱落。而混合组雌蜱的体重增加极为明显，最高增加量可达100倍以上。T-检验分析结果表明，无论是各组之间或不同叮咬天数之间，混合组与其他各组比较在体重变化上差异显著。由此得出，雌蜱在整个发育期间，必须与雄蜱交配方可完成全部吸血过程，如不进行交配，则雌蜱虽大量吸血，但并不能饱血脱落。3．雄蜱个体与个体之间蛋白组分有明显的差别，叮咬雄蜱无论是何种叮咬方式、如何配对或在何种动物上叮咬，体重小的雄蜱（此处的“体重”是指饥饿时的体重）与体重大的雄蜱相比，它们的唾液腺蛋白组分有明显差别。小个体雄蜱的唾液腺蛋白组分中，有9条蛋白带发生变化，其中分子量为49.7kDa 、35.8kDa、16kDa、12.4kDa和10.8kDa的蛋白缺失；部分小个体雄蜱的21.5kDa的蛋白量明显减少，24.9kDa和23.3kDa的蛋白量显著减少；29.3kDa的蛋白量明显增加。本研究的意义是发现了雄蜱唾液腺分泌蛋白的分子个性现象，运用蜱的分子个性这一概念，成功地解释了硬蜱吸血时的聚集现象：不同表型的蜱协同抵抗宿主的免疫反应。并在理论和实验中阐明了这一群集习性对强者与弱者个体的不同影响，及强弱共存对提高种群适应性的优势。分子个性这一概念，进一步提炼了表现型多样性的内涵，对实用领域，如抗蜱和抗蜱传病原体疫苗的开发与应用，也有指导意义。4．在对雌蜱唾液腺蛋白组分的研究中发现，在叮咬过程中，三个蛋白增加，一个蛋白减少。在混合组中，分子量为54kDa（F-A）、29.05kDa（F-B）和24.36kDa（F-C）的三个蛋白在吸血后才逐渐出现，蛋白量随着吸血天数的增加而增加，分子量为 21.08kDa(F-D)的蛋白则随着吸血程度的增加而逐渐减少，饱血后完全消失。根据文献推测，三个增加的蛋白或许为IgG结合蛋更多还原

【Abstract】 Ixodid ticks transmit the greatest variety of pathogens of any blood-sucking vector and are second only to mosquitoes in importance as vectors of human and animal disease agents. Their huge geographical range and adaptation to climatic extremes, and the diversity of hosts on which they feed, show them to be a highly successful family. The evolutionary history of the Ixodidae has been considered to relate to their host specificity. In long-established tick-host parasitic associations, some tick species developed certain mechanisms for suppressing the haemostatic and immune responses of their selected hosts. Ribeiro remarks that ticks have a highly developed ability to evade and suppress host homeostatic systems, at least for the selected host species. Other tick species which lack the specific antagonists in their saliva that help evade the host immunological response may be rapidly rejected. Moreover, Wang studies on the immunoglobulin binding protein of tick salivary gland protein in the Rhipicephalus appendiculatus showed that the male ticks could help their mates to feed.The primary antigenic source that stimulates host immunity to ticks is saliva, the secreted product of tick salivary glands. Salivary glands are the largest glands in the tick’s body. As soon as feeding starts, ticks inject the saliva into the host. Saliva from most ixodid ticks contains cement compounds that bind the tick’s mouth parts to host skin. Various enzymes and different types of bioactive molecules are also present in tick salivary glands and saliva. These components are fundamental to obtaining a successful bloodmeal. They maintain blood flow into the feeding site, antagonize host haemostatic and inflammatory mediators and help the tick evade the host’s rejection responses. Salivary glands constituents have been show to modulate host cytokine responses and reduce lymphocyte responses to T cell mitogens. Ticks can also secret paralyzing toxins via saliva that cause sickness or even death their host. The salivary gland is also the primary organ for tick-borne pathogen transmission. Furthermore, it provides a saliva-activated transmission (SAT) activity, which plays an important role in nonviraemic tick-borne virus transmission.The studies of polymorphism of salivary gland protein are major target for biology and immunology and biopharmaceutical in ticks. Some scientists are found many useful proteins from ticks salivary glands, such as histamine-binding protein, immunoglobulin-G binding protein, IL-2 binding protein, p29 gene and HL34 and 35 proteins. Now they have already isolated, cloned and expressed many proteins of ticks salivary glands. Currently the principal research is the application of recombinant expression and determination of the function of these proteins. <WP=9>In this study, we observed engorging ability of male and female ticks for differently fed and mates manner groups; analysed the polymorphism change situation of secrete proteins in ticks salivary gland; counted the egg mass weight and number and engorging time of engorged female ticks; analysed polymorphism of salivary gland for different body weight (here is unfed body weight) of male ticks; isolated and determined the amino acid sequence of immunoglobulin-G binding protein in salivary gland of male ticks. T-test methods of statistics were used to analyse the data difference of different groups and individual tick to found the biological character, and inferred the biological function (meaning). These major results were presented as following:1. Compared with same feed day whether mate or not which mates manner of male ticks, their engorged ability have not notable change. Male ticks produce and inject bioactive saliva into the feeding site, which perform their bioactive cofeeding function when they mates with the female. These bioactive matter perhaps can’t go into the systematic circulation of hosts, they can’t reach the female feeding site where they play their cofeeds function with humoral transmission, or the quantity entering of the systematic circ更多还原