Production and immunogenicity of recombinant ferric enterobactin protein (FepA)
Production and immunogenicity of recombinant ferric enterobactin protein (FepA)
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DOI：
10.1016/j.ijid.2009.12.009
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发表时间：
2010-09-01
影响因子：
8.4
通讯作者：
Rasooli, Iraj
中科院分区：
文献类型：
--
作者：
Baghal, Seyed Mehdi Larrie;Gargari, Seyed Latif Mousavi;Rasooli, Iraj
目的：大多数细菌都会产生铁铁载体复合物来获取重要元素铁。这些复合物通过通常称为 FepA（铁肠杆菌素蛋白）的受体蛋白跨外膜转运。本研究试图评价膜蛋白FepA的免疫原性，旨在抑制铁的摄取，从而保护细菌对宿主的侵袭。方法：从大肠杆菌O157:H7中扩增基因组fepA基因。将PCR产物连接至pET28a，然后在大肠杆菌BL21(DE3)中表达。将经镍-次氮基三乙酸(Ni-NTA)亲和层析纯化的重组蛋白注射到BALB/C小鼠体内诱导免疫。通过ELISA测定抗体滴度。用不同剂量的大肠杆菌 O157:H7、福氏志贺氏菌、肺炎克雷伯氏菌和伤寒沙门氏菌攻击小鼠组，以研究免疫反应。结果：表达并纯化了 85 kDa 的重组蛋白。通过注射BALB/C小鼠测定重组蛋白的免疫原性。其中产生的抗体能够有效识别并结合铁肠杆菌素结合蛋白。用较高剂量的选定细菌攻击的免疫小鼠存活下来。结论：铁肠杆菌素结合蛋白抗体的显着识别可能导致其在限制肠杆菌科细菌繁殖方面的应用。 (C) 2010 年国际传染病学会。由爱思唯尔有限公司出版。保留所有权利。
Objectives: Ferric siderophore complexes are produced by most bacteria to acquire iron, a vital element. These complexes are transported across the outer membrane by receptor proteins commonly known as FepA (ferric enterobactin protein). In this study we attempted to evaluate the immunogenicity of the membrane protein FepA, aiming at inhibition of iron uptake to protect invasion of the host by the bacterium.Methods: The genomic fepA gene was amplified from Escherichia coli O157:H7. The PCR product was ligated into pET28a and was then expressed in E. coli BL21(DE3). The recombinant protein purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was injected into BALB/C mice to induce immunity. Antibody titer was determined by ELISA. Mouse groups were challenged with various doses of E. coli O157: H7, Shigella flexneri, Klebsiella pneumoniae, and Salmonella typhi to study immune response.Results: An 85-kDa recombinant protein was expressed and purified. Immunogenicity of the recombinant protein was determined by injecting BALB/C mice. The antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein. Immunized mice challenged with higher doses of selected bacteria survived.Conclusions: Significant recognition by the antibody of ferric enterobactin binding protein may lead to its application in the restriction of Enterobacteriaceae propagation. (C) 2010 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.