兩種節(jié)肢動物過敏原的分離純化及結構與功能研究
發(fā)布時間：2019-06-20 07:41
【摘要】：昆蟲叮咬是引起速發(fā)型過敏反應的三大因素之一(其它兩類因素為食物和藥物)。通過叮咬能引起速發(fā)型過敏反應的昆蟲有蜜蜂、胡蜂、牛虻、蚊子、蟻等。被昆蟲叮咬后所引發(fā)的速發(fā)型過敏反應,由于病人未就診,醫(yī)生未報道及不同的診斷標準,其真正發(fā)生率很難計算。盡管如此,其被記載的發(fā)生率還在不斷增加。來自美國羅切斯特流行病學項目的報告,昆蟲叮咬引起的速發(fā)型過敏反應人群發(fā)生率從上世紀八十年代的十萬分之二十一增至目前的十萬分之五十,其它研究也得出相似的結果。同時這些昆蟲過敏原之間還存在不同程度的交叉過敏反應,常見的交叉過敏反應有蜜蜂蚊子交叉反應,蜜蜂胡蜂過敏原交叉反應,以及本研究所證實的胡蜂牛虻綜合征。這些昆蟲之間的交叉過敏反應使得昆蟲叮咬引起的速發(fā)型過敏反應的發(fā)生率呈倍數(shù)增長,且為這類速發(fā)型過敏反應的診治帶來較大困難。我們實驗室前期研究工作顯示牛虻的唾液腺及胡蜂的毒素富含多種具有生物活性的物質(zhì),這些生物活性物質(zhì)對牛虻從宿主獲得血餐及胡蜂捕食和自衛(wèi)有重要意義。被牛虻和胡蜂叮咬的人常會有局部的紅腫、灼痛,嚴重的可表現(xiàn)為皮膚大面積蕁麻疹,血管性水腫,支氣管痙攣,主支氣管水腫等過敏反應癥狀,甚至是過敏性休克而危及生命。但在此之前尚無牛虻過敏原的報道,對胡蜂毒素過敏原的報道也相對較少。在叮咬能引起人體過敏的昆蟲中,牛虻和胡蜂個體相對較大,分泌過敏原的唾液腺和毒腺富含唾液和毒素,在叮咬人時,可將這些唾液及毒素注入人體內(nèi),且唾液和毒素中的其它活性成分如蛋白酶、透明質(zhì)酸酶、抗凝血物質(zhì)等也有助于過敏原在機體內(nèi)的擴散,進而更易作用于免疫細胞如抗原遞呈細胞等,這些因素會導致牛虻和胡蜂叮咬引發(fā)的過敏反應癥狀更為嚴重。 在本研究中,通過解剖牛虻唾液腺及收集胡蜂毒素,整合色譜層析和免疫雜交等技術共從牛虻唾液腺和胡蜂毒素中分離純化鑒定了5種新的過敏原,它們分別是Tab y1、Vesp ma1、Vesp ma2、Vesp ma4和Vesp ma5。其中Taby1是一種分子量約為70kDa的Apyrase,它可以水解ATP生產(chǎn)AMP和雙磷酸。Vesp ma1、 Vesp ma2、Vesp ma4和Vesp ma5分別為34kDa的磷脂酶A、35kDa的透明質(zhì)酸酶、28kDa的絲氨酸蛋白酶和25kDa的抗原-5蛋白。運用Edman降解和cDNA克隆技術獲得了它們?nèi)L氨基酸序列。通過皮膚針刺實驗(Skin prick test)發(fā)現(xiàn)對牛虻過敏的病人中有約71%對Taby1呈陽性,Vesp ma5和Vesp ma2對胡蜂毒素過敏病人的陽性反應率分別為73%和80%。通過Western blot檢測發(fā)現(xiàn)Taby1對采集的37份牛虻過敏病人血清IgE的陽性率為81%, Vesp ma5和Vespa ma2對蜂中毒過敏病人血清的IgE的陽性反應率為91.0%和93.9%,對牛虻過敏病人血清IgE陽性反應率也高達86.5%和91.8%。通過Elisa competitive inhibition發(fā)現(xiàn),Tab y1可以劑量依賴的方式競爭性抑制病人血清IgE與牛虻唾液腺總蛋白的結合,最大抑制率可達42%左右,Vesp ma5和Vesp ma2對蜂毒粗樣與蜂中毒過敏病人血清IgE結合的最大抑制率分別為39.5%和39.4%,對牛虻唾液腺粗樣與牛虻過敏病人血清IgE結合的最大抑制率分別為25.3%和23.8%。嗜堿性細胞活化檢測(Basophil activation test)顯示,Taby1可以有效地誘導牛虻過敏病人外周血嗜堿性細胞CD63的表達,濃度1.0μg/ml Tab y1作用后,CD63和CCR3雙陽性細胞數(shù)是陰性對照的4.9倍。同時Taby1還具有較強的抑制ADP誘導血小板聚集活性,在濃度為230ng/ml和460ng/ml對血小板聚集的抑制率可達80%和100%。利用鼠動靜脈支路血栓模型(RAST)和角叉菜膠誘導的鼠尾血栓模型(MTT),發(fā)現(xiàn)Taby1可以呈劑量依賴地抑制血栓形成。同時,Western blot結果顯示,我們先前從牛虻唾液腺發(fā)現(xiàn)的兩類主要過敏原Taby5和Taby2與蜂毒過敏病人血清IgE陽性反應率分別可達81.8%和78.8%, Elisa competitive inhibition顯示所以Taby5和Taby2能劑量依賴地抑制蜂毒粗樣與蜂毒過敏病人血清IgE的結合。所以以上結果說明Tab y1、Vesp ma5和Vesp ma2分別是牛虻唾液腺和胡蜂毒素的主要過敏原,且Tab y2、Tab y5、Vesp ma2和Vesp ma5在胡蜂牛虻交叉過敏反應中起重要作用。 在本研究及前期研究中,我們從牛虻唾液腺和胡蜂毒素中分別鑒定了3種和4種過敏原,這些過敏原構成了牛虻唾液腺和胡蜂毒素的過敏原主要成分。這些結果豐富了我們對牛虻唾液腺及胡蜂毒素過敏原組成成分的認識,為牛虻、胡蜂及其它昆蟲叮咬所引起的速發(fā)型過敏反應的診治提供了線索和可能的治療靶標。其中這些過敏原中抗原-5蛋白和透明質(zhì)酸酶在胡蜂牛虻交叉過敏反應中起重要作用,為胡蜂牛虻綜合征的發(fā)生提供了物質(zhì)基礎。
[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.
【學位授予單位】：中國科學技術大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392
本文編號：2503050
[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.
【學位授予單位】：中國科學技術大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392
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