Expression, purification and antibody preparation of flagellin FlaA from Vibrio alginolyticus strain HY9901.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Liang
H Y
HY
South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
Xia
L Q
LQ
Wu
Z H
ZH
Jian
J C
JC
Lu
Y S
YS
D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry / Q000276:immunology*; D001428:Bacterial Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005408:Flagellin / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D015870:Gene Expression; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D044149:Vibrio alginolyticus / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D005408:Flagellin; C067138:flaA protein, bacteria