副溶血性弧菌vanM基因原核表达及酰基高丝氨酸内酯信号分子鉴定研究

Research on Prokaryotic Expression of Vibrio parahaemolyticus vanM Gene and N- acyl-homoserine Lactone Identification

摘要:

从副溶血性弧菌ATCC33847扩增vanM基因，连接到pMD19-T载体进行克隆测序和序列比对；将测序正确的vanM序列经NdeI和EcoRI酶切后连接到pET22b；以IPTG诱导vanM基因在BL21（DE3）中表达；利用acyl-HSL报告菌株KYC55检测ATCC33847和带有vanM基因的大肠杆菌的acyl-HSL活性；萃取VanM合成的acyl-HSL信号分子，经HPLC-MS测试后与标准品进行对比分析。成功测序了ATCC33847 vanM基因，与鳗弧菌vanM基因序列相似性达到57%；并构建了pET22b-vanM表达质粒，以0.6 mmol/L IPTG诱导BL21（DE3）表达系统时VanM融合蛋白表达量最大；经过KYC55检测ATCC33847和带有pET22b-vanM的大肠杆菌能够产生acyl-HSL活性；HPLC-MS分析显示，ATCC33847和携带pET22b-vanM的BL21(DE3)萃取物中含有3-Hydroxybutanoyl-HSL (3-OH-C4-HSL)和3-Hydroxydecanoyl-HSL (3-OH-C10-HSL)信号分子。本研究克隆表达了副溶血性弧菌vanM，首次证明了副溶血性弧菌利用VanM信号分子合成酶合成acyl-HSL信号分子3-OH-C4-HSL和3-OH-C10-HSL。

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.