Purification and molecular cloning of and immunization with Ancylostoma ceylanicum excretory-secretory protein 2, an immunoreactive protein produced by adult hookworms
Purification and molecular cloning of and immunization with Ancylostoma ceylanicum excretory-secretory protein 2, an immunoreactive protein produced by adult hookworms
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DOI：
10.1128/iai.72.4.2203-2213.2004
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发表时间：
2004-04-01
影响因子：
3.1
通讯作者：
Cappello, M
中科院分区：
文献类型：
--
作者：
Bungiro, RD;Solis, CV;Cappello, M
钩虫仍然是全球发病率的主要因素，针对这些吸血寄生虫的疫苗接种可能是传统控制方法的一种有吸引力的补充。在这里，我们描述了克隆锡兰钩虫排泄分泌蛋白2（AceES-2），一种新的免疫反应性蛋白质产生的成虫。通过反相高压液相色谱法从排泄分泌（ES）产物中纯化天然AceES-2，进行氨基末端测序，并通过使用逆转录-PCR从成虫RNA中克隆AceES-2。AceES-2cDNA预测其成熟蛋白由102个氨基酸组成，分子量为11.66kDa。Western免疫印迹和酶联免疫吸附试验表明，重组AceES-2（rAceES-2）与A.感染锡兰菌的仓鼠用吸附到明矾上的天然ES产物免疫仓鼠诱导识别rAceES-2的抗体，而rAceES-2-明矾疫苗接种导致与ES产物中的单个蛋白条带反应的抗体，该蛋白条带非常接近天然分子的预测大小。与接受正常血清的对照组相比，用超免疫兔抗rAceES-2血清被动免疫的感染仓鼠表现出更快和完全的贫血恢复。rAceES-2的口服免疫与攻击后贫血显著减少相关，结果类似于口服免疫可溶性钩虫提取物的仓鼠中观察到的结果（后者动物也对体重减轻有抗性）。这些数据表明，AceES-2在宿主-寄生虫相互作用中起着重要作用，针对该蛋白质的疫苗接种可能是控制钩虫贫血的有用策略。
Hookworms remain major agents of global morbidity, and vaccination against these bloodfeeding parasites may be an attractive complement to conventional control methods. Here we describe the cloning of Ancylostoma ceylanicum excretdry-secretory protein 2 (AceES-2), a novel immunoreactive protein produced by adult worms. Native AceES-2 was purified from excretory-secretory (ES) products by reverse-phase high-pressure liquid chromatography, subjected to amino-terminal sequencing, and cloned from adult worm RNA by using reverse transcription-PCR. The translated AceES-2 cDNA predicts that the mature protein consists of 102 amino acids and has a molecular mass of 11.66 kDa. Western immunoblot and enzyme-linked immunosorbent assay analyses demonstrated that recombinant AceES-2 (rAceES-2) reacted strongly with antibodies from A. ceylanicum-infected hamsters. Immunization of hamsters with native ES products adsorbed to alum induced antibodies that recognized rAceES-2, while rAceES-2-alum vaccination resulted in antibodies that reacted with a single protein band in ES products that closely approximated the size predicted for the native molecule. Infected hamsters that were passively immunized with hyperimmune rabbit anti-rAceES-2 serum exhibited more rapid and complete recovery from anemia than controls that received normal serum. Oral immunization with rAceES-2 was associated with significantly reduced anemia upon challenge, an outcome similar to the outcome observed in hamsters that were orally vaccinated with soluble hookworm extract (the latter animals were also resistant to weight loss). These data suggest that AceES-2 plays an important role in the host-parasite interaction and that vaccination against this protein may represent a useful strategy for controlling hookworm anemia.