天然生物活性N-酰基高丝氨酸内酯小分子抗原的化学合成及其抗体生成初探

目的人工合成N-酰基高丝氨酸内酯(N-acylated homoserine lactones,N-aHSLs)完全抗原并制备相应单抗，建立N-aHSLs免疫检测的新方法及海洋弧菌N-aHSLs动态监测。方法以月桂酸和高丝氨酸内酯为主要原料，通过多步系列化学反应人工合成N 十二酰高丝氨酸内酯(N-lauroyl-homoserine lactone,N-C12-HSL)及含羧基活性交联基团的半抗原衍生物12-羧基-N-十二酰高丝氨酸内酯（12-carboxyl-N-decanoyl-L-homoserine lactone,12-CD-LHL），用1H-磁共振、高压/效液相色谱仪和液相色谱 质谱联用方法确证合成产物的结构，并用生物感应法检测合成N-aHSLs的天然生物活性；用N-羟基琥珀酰亚胺活泼酯法将12-CD-LHL半抗原衍生物连接到载体蛋白；将N-C12-HSL人工完全抗原通过传统单抗制备技术，获得抗N-aHSLs杂交瘤细胞株，用酶联免疫吸附测定法鉴定其特异性和效价。结果质谱、磁共振氢谱分析结果与理论预测相同，成功合成目标产物N-C12-HSL和12-CD-LHL半抗原衍生物；所合成的N-aHSLs能够分别激活生物感应菌株大肠杆菌MG4和紫色色杆菌026并使之显色；紫外吸收光谱扫描结果显示，半抗原已分别与牛血清白蛋白和卵清蛋白发生偶联，成功制备完全抗原；共获得3株抗N-C12-HSL单抗，均具有良好的免疫反应性。结论N-aHSLs完全抗原和相应的单抗制备为N-aHSLs动态监测和后续研究奠定了基础。

Chemosynthesis of N-acylated homoserine lactones hapten derivant and the preliminary study of its antibody formation

Objective To synthesize the artificial holoantigen and corresponding monoclonal antibodies of N-acylated-homoserine lactones (N-aHSLs), and to provide basis for establishment of a new immunodetection and monitoring method of N-aHSLs. MethodsLauric acid and homoserine lactone were used as the starting materials to synthesize N-lauroyl-homoserine lactone (N-C12-HSL) and the hapten derivant of 12-carboxyl-N-decanoyl-L-homoserine lactone (12-CD-LHL) with chemical crosslinking active group by serial reactions. The target products were characterized by 1H nuc lear magnetic resonance (1H-NMR), high pressure/performance liquid chromatography (HPLC) and liquid chromatograph mass spectrometer (LC-MS). The natural bioactivity of N-aHSLs was detected by biological response method. Holoantigen of N-C12-HSL was synthesized by the method of active ester. BALB/c mice was immunized with N-C12-HSL holoantigen, and hybridoma cells against N-C12-HSL were produced by cell fusion technique. The specificity and titers of monoclonal antibody against N-C12-HSL were screened by enzyme linked immunosorbent assay. ResultsThe 1H-NMR, HPLC and LC-MS results revealed that the obtained compound were the target product of N-C12-HSL and 12-CD-LHL, just as the prediction. The synthetical N-aHSLs could activate the biological response strains of escherichia coli and chromobacterium violaceum 026 seperately and color the strains. The ultraviolet absorption spectrum results revealed that hapten N-C12-HSL had crosslinked with bovine serum albumin and ovalbumin, N-C12-HSL holoantigen had been prepared well. Three strains of hybridoma against N-C12-HSL were obtained, and the identification results indicated which all have favourable immunoreactivity. ConclusionThe preparation of N-aHSLs whole antigen and corresponding monoclonal antibody laid the foundation for N-aHSLs dynamic monitoring and subsequent research.