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首页> 外文期刊>Applied Microbiology and Biotechnology >The development of a flagellin surface display expression system in a moderate thermophile, Bacillus halodurans Alk36
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The development of a flagellin surface display expression system in a moderate thermophile, Bacillus halodurans Alk36

机译：中度嗜热嗜热芽孢杆菌Alk36鞭毛蛋白表面展示表达系统的开发

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摘要

This study relates to the development of an alkaliphilic, thermotolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium continuously over-produces flagellin. To harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene, which codes for flagellin, was inactivated on the chromosome giving rise to the B. halodurans BhFC01 mutant. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-1 subtype C gp120 epitope were, respectively, incorporated into this site as in-frame fusions. The peptides were found to be successfully displayed on the cell surface and functional through metal binding and immunological studies, respectively.
机译：这项研究涉及开发一种嗜碱，耐热，革兰氏阳性分离株，嗜盐芽孢杆菌Alk36，用于嵌合基因产物的过量生产和表面展示。这种细菌不断产生鞭毛蛋白。为了利用这种能力，为该菌株开发了关键的遗传工具，例如基因靶向灭活。编码鞭毛蛋白的hag基因在染色体上失活，从而产生了嗜盐芽孢杆菌BhFC01突变体。将多接头作为框内嵌合鞭毛蛋白夹心融合物插入，以鉴定与鞭毛蛋白可变区相对应的允许插入位点。对于这些构建体，评估鞭毛蛋白的表达和运动性。确定了两个位点可能在鞭毛蛋白基因中插入了肽，其中一个产生了功能性鞭毛并且能够将运动性表型恢复为非运动性突变体。分别将编码聚组氨酸肽和HIV-1亚型C gp120表位的肽作为框内融合物掺入该位点。分别通过金属结合和免疫学研究，发现该肽成功地展示在细胞表面并具有功能。

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