禽坏死性肠炎候选嵌合疫苗的设计与表达

A. Rostami, F. Goshadrou, R. P. Langroudi, S. Z. Bathaie, A. Riazi, J. Amani, G. Ahmadian
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引用次数: 16

摘要

坏死性肠炎是由产气荚膜梭菌引起的一种重要的家禽疾病。据报道,细菌释放多种毒素,其中NetB、α毒素和TpeL在疾病的致病性和/或严重程度中发挥重要作用。本研究利用生物信息学工具分析了梭菌毒素NetB、α毒素和TpeL的序列,以确定具有高免疫原性因子的蛋白结构域。设计并评价了由三种毒素免疫原区组成的几种嵌合三价蛋白。分开的区域用刚性连接件粘合在一起。基于模拟的三级结构,选择合适的组合并在细菌宿主(大肠杆菌)中表达并成功纯化。western blotting进一步验证了嵌合蛋白的表达。免疫血清识别每个嵌合蛋白亚基的能力也被检测。利用圆二色性对预测的嵌合蛋白二级结构进行评价。体外效价试验表明,嵌合蛋白免疫的兔血清能够部分中和α毒素,因此该构建物可作为产气荚膜荚膜原细菌的疫苗。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Design and expression of a chimeric vaccine candidate for avian necrotic enteritis
Necrotic enteritis is an economically important disease of poultry mainly caused by Clostridium perfringens. The bacteria release multiple toxins of which NetB, alpha toxin and TpeL have been reported to play important roles in pathogenicity and/or severity of the disease. In this study, the sequence of clostridial toxins NetB, alpha toxin and TpeL were analyzed using bioinformatics tools to determine protein domains with high immunogenicity factor. Several chimeric trivalent proteins consisting of the immunogenic regions of the three toxins were designed and evaluated. The separate regions were fused together using rigid linkers. Based on a modeled tertiary structure, a proper combination was selected and expressed in a bacterial host (Escherichia coli) and successfully purified. The expression of the chimeric protein was further verified by western blotting. The ability of the immunized serum in recognizing each individual subunit of the chimeric protein was also examined. Circular dichroism was used to evaluate the predicted secondary structure of the chimeric protein. In vitro potency test demonstrated that the serum from a rabbit immunized with the chimeric protein is able to partially neutralize Alpha toxin, hence the construct can potentially be used as a vaccine against C. perfringens.
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