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Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901
Liang, HY; Xia, LQ; Wu, ZH; Jian, JC; Lu, YS; wuzh@gdou.edu.cn; jianjc@gmail.com
2010
发表期刊LETTERS IN APPLIED MICROBIOLOGY
ISSN0266-8254
卷号50期号:2页码:181-186
摘要Aims: 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. Methods and Results: 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). Conclusions: 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. Significance and Impact of the Study: 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.
部门归属[Liang, H. Y.; Xia, L. Q.; Wu, Z. H.; Jian, J. C.; Lu, Y. S.] Guangdong Ocean Univ, Coll Fisheries, Zhanjiang City 524025, Guangdong, Peoples R China; [Liang, H. Y.] Chinese Acad Sci, S China Sea Inst Oceanol, Guangzhou, Guangdong, Peoples R China; [Liang, H. Y.] Chinese Acad Sci, Grad Sch, Beijing, Peoples R China; [Liang, H. Y.; Wu, Z. H.; Jian, J. C.; Lu, Y. S.] Guangdong Prov Key Lab Pathogen Biol & Epidemiol, Zhanjiang, Peoples R China; [Liang, H. Y.; Wu, Z. H.; Jian, J. C.; Lu, Y. S.] Guangdong Key Lab Control Dis Aquat Econ Anim, Zhanjiang, Peoples R China
关键词Antibody Preparation Expression Flagellin Flaa Gene Immunogold Electron Microscopy Purification Vibrio Alginolyticus
学科领域Biotechnology & Applied Microbiology ; Microbiology
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收录类别sci
资助项目海洋生物
WOS记录号WOS:000273316800008
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文献类型期刊论文
条目标识符http://ir.scsio.ac.cn/handle/344004/5442
专题海洋生物
通讯作者wuzh@gdou.edu.cn; jianjc@gmail.com
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Liang, HY,Xia, LQ,Wu, ZH,et al. Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901[J]. LETTERS IN APPLIED MICROBIOLOGY,2010,50(2):181-186.
APA Liang, HY.,Xia, LQ.,Wu, ZH.,Jian, JC.,Lu, YS.,...&jianjc@gmail.com.(2010).Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901.LETTERS IN APPLIED MICROBIOLOGY,50(2),181-186.
MLA Liang, HY,et al."Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901".LETTERS IN APPLIED MICROBIOLOGY 50.2(2010):181-186.
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