AMBLYOMMA-AMERICANUM - PHYSIOCHEMICAL ISOLATION OF A PROTEIN DERIVED FROM THE TICK SALIVARY-GLAND THAT IS CAPABLE OF INDUCING IMMUNE RESISTANCE IN GUINEA-PIGS
AMBLYOMMA-AMERICANUM - PHYSIOCHEMICAL ISOLATION OF A PROTEIN DERIVED FROM THE TICK SALIVARY-GLAND THAT IS CAPABLE OF INDUCING IMMUNE RESISTANCE IN GUINEA-PIGS
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DOI：
10.1016/0014-4894(86)90006-8
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发表时间：
1986-08-01
影响因子：
2.1
通讯作者：
ASKENASE, PW
中科院分区：
文献类型：
--
作者：
BROWN, SJ;ASKENASE, PW
通过理化（凝胶过滤和离子交换色谱）和免疫化学豚鼠IgG 1（抗蜱免疫亲和柱）技术分析美洲钝眼蜱唾液腺衍生粗蛋白，以确定是否存在诱导宿主免疫抗性反应的抗原。粗唾液腺抗原的凝胶过滤（G-75 Sephadex）和离子交换（二乙基氨基乙基纤维素）色谱法得到多个组分，但当用于静脉注射0.05%伊文思蓝染料的蜱致敏动物的皮肤试验时，每个程序中只有一个组分诱导显著的皮肤过敏性变蓝反应。唾液腺抗原（200 ng）用0.2 M Na 2CO 3，pH 11.3从免疫亲和柱洗脱，并用不完全弗氏佐剂乳化，赋予显著水平的蜱排斥（24%，P < 0.001）与对照组相比，但低于（P < 0.01）由粗唾液腺抗原（380 μ g）赋予的免疫水平。免疫亲和纯化的唾液腺抗原的免疫剂量是粗抗原制剂剂量的1/1900，代表99.9%的纯化。此外，来自用唾液腺抗原免疫的动物的饱食蜱与来自未处理动物的蜱相比表现出重量的显著降低（P < 0.001）。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的125 I标记的蛋白质在Na 2CO 3凝胶和皮肤反应的凝胶过滤和离子交换色谱的馏分，免疫沉淀后与豚鼠IgG 1抗体转移阻力的蜱，揭示了存在的20 kDa的重量蛋白质先前报告的抗原负责诱导宿主耐药性。这些研究提供了纯化重要蜱蛋白的理化和免疫化学方法，该蛋白（1）在蜱致敏豚鼠中诱导皮肤反应，（2）被蜱抗体识别，最重要的是，（3）能够免疫未处理豚鼠对抗蜱攻击。
Crude salivary gland derived proteins from Amblyomma americanum ticks were analyzed by physiochemical (gel filtration and ion exchange chromatography) and immunochemical guinea pig IgG1 (anti-tick immunoaffinity column) techniques for the presence of antigens responsible for the induction of host immune resistance responses. Gel filtration (G-75 Sephadex) and ion exchange (diethylaminoethyl cellulose) chromatography of crude salivary gland antigen yielded multiple fractions, but only one fraction from each procedure induced significant cutaneous anaphylaxis bluing reactions when used for skin tests in tick sensitized animals treated intravenously with 0.05% Evans blue dye. Salivary gland antigen (200 ng) eluted from the immunoaffinity column by 0.2 M Na2CO3, pH 11.3, and emulsified with incomplete Freund''s adjuvant conferred a significant level of tick rejection (24%, P < 0.001) on naive guinea pigs compared with that seen in controls, but less than (P < 0.01) the level of immunity conferred by crude salivary gland antigen (380 .mu.g). The immunizing dose of immunoaffinity purified salivary gland antigen was 1/1900 the dose of the crude antigen preparation representing 99.9% purification. Furthermore, engorged ticks from animals immunized with salivary gland antigen exhibited a significant decrease (P < 0.001) in weight compared with ticks from naive animals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I labeled proteins in the Na2CO3 eluate and the skin reactive fraction from gel filtration and ion-exchange chromatography, after immunoprecipitation with a guinea pig IgG1 antibody to the tick that transferred resistance, revealed the presence of a 20 kDa weight protein reported previously to be the antigen responsible for the induction of host resistance. These studies present physiochemical and immunochemical procedures for the purification of an important tick protein that (1) induces skin reactions in tick sensitized guinea pigs, (2) is recognized by antibody to the tick, and most importantly, (3) is capable of immunizing naive guinea pigs against tick challenge.