| Vibrio parahaemolyticus is a gram-negative, halophilic bacterium which is widely distributed over coastal area, and is also a food-borne pathogen causing human acute gastroenteritis and reactive arthritis. V. parahaemolyticus possesses dual flagellar systems adapted for movement under different circumstances. Flagellar protein can be utilized to produce antibodies due to its high immunogenicity. In this work, recombinant bacteria expressing flaE gene of V. parahaemolyticus were constructed successfully, and target protein was obtained after IPTG induction and affinity chromatography to produce polyclonal antibodies. All this achievement would be useful for preparing immunomagnetic beads, and for developing the technique in rapid enrichment and detection of V. parahaemolyticus.Primers were designed and synthesized according to the sequence of flaE gene of V. parahaemolyticus BB22 published in GeneBank (U12817.2). V. parahaemolyticus flaE gene was amplified by PCR with high-fidelity polymerase from ATCC17802, subsequently inserted in pMD 19-T vector and transformed into E. coli DH5a. Positive strains are selected by Amp, blue-white selection and colony PCR. According to sequencing result, the cloned gene is 1122bp, and 98% identical to flaE gene of BB22. After double restriction endonucleases digestion and ligation, the recombinant express plasmid pET22b-flaE was constructed and then transformed into E.coli BL21(DE3).FlaE protein was induced under 37℃with 0.8mM IPTG for 3 hours. SDS-PAGE analysis suggested that the molecular weight of the expressed protein is 40kDa which correspond to the DNASTAR calculation, and the protein mostly lied in inclusion bodies. The expressed protein fused with His tag was purified by the method of affinity chromatography and then quantitated by BCA. Purified protein was misced with adjuvant to immune New Zealand white rabbit. The result of ELISA showed that the highest titer of anti-serum was 1:204800. |
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