South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
Lett Appl Microbiol. 2010 Feb;50(2):181-6. doi: 10.1111/j.1472-765X.2009.02768.x. Epub 2009 Nov 5.
The main aims of this study were to clone and express flagellin flaA gene from Vibrio alginolyticus strain HY9901, also to prepare mouse anti-FlaA polyclonal antibody for future pathogen or vaccine study.
The full-length flaA gene was amplified by PCR with designed primers. The open reading frame of flaA gene contains 1131 bp, and its putative protein consists of 376 amino acid residues. Alignment analysis indicated that the FlaA protein was highly conserved. SDS-PAGE indicated that the FlaA protein was successfully expressed in Escherichia coli BL21 (DE3). Then, the recombinant FlaA protein was purified by affinity chromatography, and the mouse anti-FlaA serum was produced. The expression of flaA gene was verified by various immunological methods, including western blotting, enzyme-linked immunosorbent assay (ELISA) and immunogold electron microscopy (IEM).
Flagellin flaA gene was cloned and identified from V. alginolyticus HY9901, the recombinant FlaA protein was expressed and purified, and high-titre FlaA protein-specific antibody was produced. Western blot analysis revealed that the prepared antiserum not only specifically react to FlaA fusion protein, but also to natural FlaA protein of V. alginolyticus. The expressed FlaA protein was demonstrated, for the first time, as the component of flagella from V. alginolyticus by IEM.
This study may offer important insights into the pathogenesis of V. alginolyticus, provide a base for further studies on the diagnosis and evaluation that whether the FlaA protein could be used as an effective vaccine candidate against infection by V. alginolyticus and other Vibrio species. Additionally, the purified FlaA protein and polyclonal antibody can be used for further functional and structural studies.
本研究的主要目的是克隆和表达从哈氏弧菌 HY9901 中提取的鞭毛蛋白 flaA 基因，并制备小鼠抗 FlaA 多克隆抗体，为未来的病原体或疫苗研究做准备。
通过设计引物，用 PCR 扩增全长 flaA 基因。flaA 基因的开放阅读框包含 1131bp，其推测蛋白由 376 个氨基酸残基组成。序列比对分析表明 FlaA 蛋白高度保守。SDS-PAGE 表明 FlaA 蛋白在大肠杆菌 BL21（DE3）中成功表达。然后，通过亲和层析纯化重组 FlaA 蛋白，并制备小鼠抗 FlaA 血清。通过各种免疫学方法，包括 Western blot、酶联免疫吸附试验（ELISA）和免疫金电子显微镜（IEM）验证 flaA 基因的表达。
从哈氏弧菌 HY9901 中克隆并鉴定了鞭毛蛋白 flaA 基因，表达并纯化了重组 FlaA 蛋白，制备了高滴度 FlaA 蛋白特异性抗体。Western blot 分析表明，制备的抗血清不仅特异性地与 FlaA 融合蛋白反应，还与哈氏弧菌的天然 FlaA 蛋白反应。首次通过 IEM 证明表达的 FlaA 蛋白是哈氏弧菌鞭毛的组成部分。
本研究可能为哈氏弧菌的发病机制提供重要的见解，为进一步研究 FlaA 蛋白作为哈氏弧菌及其他弧菌感染的有效疫苗候选物的诊断和评估提供基础。此外，纯化的 FlaA 蛋白和多克隆抗体可用于进一步的功能和结构研究。
