Research on Prokaryotic Expression of Vibrio parahaemolyticus vanM Gene and N- acyl-homoserine Lactone Identification
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Abstract:
The vanM sequence was amplified for V. parahaemolyticus ATCC33847 genome and inserted into pMD19-T vector for clone sequencing and sequence alignment. The correct vanM fragment was double-digested by NdeI and EcoRI and ligated into the pET22b vector for gene expression. IPTG was used to induce vanM prokaryotic expression. An acyl-HSL bio-reporter strain KYC55 was used to detect the acyl-HSL activities of ATCC33847 and Escherichia coli carrying the vanM gene. HPLC-MS assay was performed to identify vanM-associated acyl-HSL molecules in extracts from ATCC33847 and Escherichia coli. The ATCC33847 vanM sequence was aligned with that of V. anguillarum vanM, with the similarity of 57%. The expression vector pET22b-vanM was constructed and induced to produce the highest level of VanM protein by induction with 0.6mmol/L IPTG in BL21(DE3). ATCC33847 and BL21(DE3) containing pET22b-vanM presented an acyl-HSL activity, which was detected by KYC55. HPLC-MS results indicated that both 3-Hydroxybutanoyl-HSL (3-OH-C4-HSL) and 3-Hydroxydecanoyl-HSL (3-OH-C10-HSL) were detected in extracts from ATCC33847 and BL21(DE3)(pET22b-vanM). In this study, the prokaryotic expression of V. parahaemolyticus vanM confirmed that V. parahaemolyticus vanM was responsible for the synthesis of V. parahaemolyticus quorum sensing signals, 3-OH-C4-HSL and 3-OH-C10-HSL.
