两种节肢动物过敏原的分离纯化及结构与功能研究

发布时间：2019-06-20 07:41

【摘要】：昆虫叮咬是引起速发型过敏反应的三大因素之一(其它两类因素为食物和药物)。通过叮咬能引起速发型过敏反应的昆虫有蜜蜂、胡蜂、牛虻、蚊子、蚁等。被昆虫叮咬后所引发的速发型过敏反应,由于病人未就诊,医生未报道及不同的诊断标准,其真正发生率很难计算。尽管如此,其被记载的发生率还在不断增加。来自美国罗切斯特流行病学项目的报告,昆虫叮咬引起的速发型过敏反应人群发生率从上世纪八十年代的十万分之二十一增至目前的十万分之五十,其它研究也得出相似的结果。同时这些昆虫过敏原之间还存在不同程度的交叉过敏反应,常见的交叉过敏反应有蜜蜂蚊子交叉反应,蜜蜂胡蜂过敏原交叉反应,以及本研究所证实的胡蜂牛虻综合征。这些昆虫之间的交叉过敏反应使得昆虫叮咬引起的速发型过敏反应的发生率呈倍数增长,且为这类速发型过敏反应的诊治带来较大困难。我们实验室前期研究工作显示牛虻的唾液腺及胡蜂的毒素富含多种具有生物活性的物质,这些生物活性物质对牛虻从宿主获得血餐及胡蜂捕食和自卫有重要意义。被牛虻和胡蜂叮咬的人常会有局部的红肿、灼痛,严重的可表现为皮肤大面积荨麻疹,血管性水肿,支气管痉挛,主支气管水肿等过敏反应症状,甚至是过敏性休克而危及生命。但在此之前尚无牛虻过敏原的报道,对胡蜂毒素过敏原的报道也相对较少。在叮咬能引起人体过敏的昆虫中,牛虻和胡蜂个体相对较大,分泌过敏原的唾液腺和毒腺富含唾液和毒素,在叮咬人时,可将这些唾液及毒素注入人体内,且唾液和毒素中的其它活性成分如蛋白酶、透明质酸酶、抗凝血物质等也有助于过敏原在机体内的扩散,进而更易作用于免疫细胞如抗原递呈细胞等,这些因素会导致牛虻和胡蜂叮咬引发的过敏反应症状更为严重。在本研究中,通过解剖牛虻唾液腺及收集胡蜂毒素,整合色谱层析和免疫杂交等技术共从牛虻唾液腺和胡蜂毒素中分离纯化鉴定了5种新的过敏原,它们分别是Tab y1、Vesp ma1、Vesp ma2、Vesp ma4和Vesp ma5。其中Taby1是一种分子量约为70kDa的Apyrase,它可以水解ATP生产AMP和双磷酸。Vesp ma1、 Vesp ma2、Vesp ma4和Vesp ma5分别为34kDa的磷脂酶A、35kDa的透明质酸酶、28kDa的丝氨酸蛋白酶和25kDa的抗原-5蛋白。运用Edman降解和cDNA克隆技术获得了它们全长氨基酸序列。通过皮肤针刺实验(Skin prick test)发现对牛虻过敏的病人中有约71%对Taby1呈阳性,Vesp ma5和Vesp ma2对胡蜂毒素过敏病人的阳性反应率分别为73%和80%。通过Western blot检测发现Taby1对采集的37份牛虻过敏病人血清IgE的阳性率为81%, Vesp ma5和Vespa ma2对蜂中毒过敏病人血清的IgE的阳性反应率为91.0%和93.9%,对牛虻过敏病人血清IgE阳性反应率也高达86.5%和91.8%。通过Elisa competitive inhibition发现,Tab y1可以剂量依赖的方式竞争性抑制病人血清IgE与牛虻唾液腺总蛋白的结合,最大抑制率可达42%左右,Vesp ma5和Vesp ma2对蜂毒粗样与蜂中毒过敏病人血清IgE结合的最大抑制率分别为39.5%和39.4%,对牛虻唾液腺粗样与牛虻过敏病人血清IgE结合的最大抑制率分别为25.3%和23.8%。嗜碱性细胞活化检测(Basophil activation test)显示,Taby1可以有效地诱导牛虻过敏病人外周血嗜碱性细胞CD63的表达,浓度1.0μg/ml Tab y1作用后,CD63和CCR3双阳性细胞数是阴性对照的4.9倍。同时Taby1还具有较强的抑制ADP诱导血小板聚集活性,在浓度为230ng/ml和460ng/ml对血小板聚集的抑制率可达80%和100%。利用鼠动静脉支路血栓模型(RAST)和角叉菜胶诱导的鼠尾血栓模型(MTT),发现Taby1可以呈剂量依赖地抑制血栓形成。同时,Western blot结果显示,我们先前从牛虻唾液腺发现的两类主要过敏原Taby5和Taby2与蜂毒过敏病人血清IgE阳性反应率分别可达81.8%和78.8%, Elisa competitive inhibition显示所以Taby5和Taby2能剂量依赖地抑制蜂毒粗样与蜂毒过敏病人血清IgE的结合。所以以上结果说明Tab y1、Vesp ma5和Vesp ma2分别是牛虻唾液腺和胡蜂毒素的主要过敏原,且Tab y2、Tab y5、Vesp ma2和Vesp ma5在胡蜂牛虻交叉过敏反应中起重要作用。在本研究及前期研究中,我们从牛虻唾液腺和胡蜂毒素中分别鉴定了3种和4种过敏原,这些过敏原构成了牛虻唾液腺和胡蜂毒素的过敏原主要成分。这些结果丰富了我们对牛虻唾液腺及胡蜂毒素过敏原组成成分的认识,为牛虻、胡蜂及其它昆虫叮咬所引起的速发型过敏反应的诊治提供了线索和可能的治疗靶标。其中这些过敏原中抗原-5蛋白和透明质酸酶在胡蜂牛虻交叉过敏反应中起重要作用,为胡蜂牛虻综合征的发生提供了物质基础。
[Abstract]:The insect bite is one of the three main factors that cause an allergic reaction to a quick-onset type (the other two factors are food and medicine). An insect that can cause an allergic reaction by biting can be a bee, a bee, a cow, a mosquito, an ant, and the like. The rate of allergic reaction caused by insect bite is difficult to calculate due to the fact that the patient has not seen a doctor, the doctor has not reported and different diagnostic criteria. Nevertheless, the rate of the record is increasing. The incidence of rapid-type allergic reaction caused by insect bites has increased from the twenty-first century in the 1980s to fifty of the current ten-hundred and fifty-five, and other studies have also yielded similar results. At the same time, there is a different degree of cross-allergic reaction between these insect allergens. The common cross-allergic reaction has the cross-reaction of the bee-mosquito, the cross-reaction of the bee-bee-bee's allergen, and the syndrome of the bee-bee-cow syndrome confirmed by the institute. The cross-allergic reaction between these insects has led to a multiple increase in the rate of rapid-type allergic reactions caused by insect bites, and has brought about a great difficulty in the diagnosis and treatment of this type of anaphylaxis. Our laboratory studies have shown that the toxin of the salivary glands and wasps of the cow is rich in a variety of biologically active substances, which are of great significance to the acquisition of blood and the predation and self-defence of the cattle from the host. The people bitten by the cow and the bee are often red, swollen and burning, and the serious symptoms can be skin large-area urticaria, angioedema, bronchospasm, main bronchial edema and other allergic reaction symptoms, even anaphylactic shock and life-threatening. However, there were no reports of bovine allergen, and there were also relatively few reports on the allergen of the bee venom. in an insect that can cause an allergic reaction to a human body, that individual of the cow and the bee is relatively large, the salivary gland and the toxic gland that secrete the allergen are rich in saliva and toxin, and when the bite is a person, the saliva and the toxin can be injected into the human body, and other active components of the saliva and the toxin such as the protease, Hyaluronic acid enzymes, anticoagulated substances, and the like also contribute to the spread of the allergen in the body, and are more likely to act on immune cells, such as antigen-presenting cells, and the like, which can lead to more severe allergic reactions caused by the bite of the cattle and the bee. In this study, five new allergens were isolated and purified from the salivary glands of the cattle and the wasp toxin by dissecting the salivary glands of the bovine and the collection of the wasps toxin, the whole-in-the-the-column chromatography and the immunohybridization. They were Tab 1, Vespma1, Vespma2, Vespma4 and Vespma, respectively. 5. Tabyl is an apyrase with a molecular weight of about 70 kDa, which can hydrolyze ATP to produce AMP and diphosphorus Acid. Vespma1, Vespma2, Vespma4 and Vespma5 are respectively 34 kDa phospholipase A,35 kDa hyaluronidase,28 kDa serine protease and 25 kDa antigen-5 egg The full-length amino acid sequence was obtained by Edman degradation and cDNA cloning. The positive rates of positive, Vespma5, and Vespma2 in patients with allergic reactions were 73% and 80%, respectively. By Western blot, the positive rate of serum IgE in 37 patients with allergic reaction was 81%, and that of Vespma5 and Vespa ma2 was 91.0% and 93.9%, and the serum IgE positive rate was 86.5% and 91.8% in patients with allergic reaction. %. Tab y1 can competitively inhibit the binding of serum IgE to the total protein of bovine salivary gland in a dose-dependent manner, with a maximum inhibition rate of up to 42%. The maximum inhibitory rate of the combination of right, Vespma5 and Vespma2 on the serum IgE in the patients with bee venom and bee-poisoned patients was 39.5% and 39.4%, respectively. The maximum inhibition rate of serum IgE binding in the salivary gland of the cattle and the patients with allergic reaction was 25.3% and 23.8, respectively. %. The activation test of basophilic cells showed that Tabyl could effectively induce the expression of CD63 in peripheral blood of patients with allergic reaction. After the concentration of 1.0. mu.g/ ml of Tab 1, the number of CD63 and CCR3 double positive cells was 4.9 in negative control. The inhibition rate of platelet aggregation was up to 80% and 100 ng/ ml at the concentration of 230 ng/ ml and 460 ng/ ml. %. Tabyl was found to be in a dose-dependent manner to inhibit thrombosis, using a rat tail thrombus model (RAST) and a carrageenan-induced rat tail thrombus model (MTT). The results of Western blot showed that the serum IgE-positive rates of the two main allergens, Taby5 and Taby2, which were found in the salivary glands of the cattle, were 81.8% and 78.8, respectively. %, Elisa, and Taby2 show that Taby5 and Taby2 can be used to dose-dependently inhibit the binding of the bee venom and the serum IgE in the patients with bee venom. The results indicated that Tab y1, Vespma5, and Vespma2 were the main allergens of the salivary glands of the cattle, and the Vespma2, respectively, and the Tab y2, Tab y5, Vespma2 and Vespma5 play an important role in the cross-reaction of the cross-section of the bovis. In this study and in that early study, we identified three and four allergens from the salivary gland of the cow and the bee toxin, which constitute the main allergen of the salivary gland of the cow and the bee toxin. These results enrich our understanding of the composition of the components of the salivary gland of the cow and the allergen of the bee toxin, and provide clues and possible treatment for the diagnosis and treatment of the allergic reaction caused by the biting of the cow, the bee and other insects. The antigen-5 protein and the hyaluronidase in these allergens play an important role in the cross-allergic reaction of the bumblebee cattle, and provides an object for the occurrence of the euphorbia syndrome.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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