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| 吡虫啉人工半抗原合成方法的优化 |
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钟艺煊1,3,王彦红1,3,杨成根1,2,邹辉1,3,顾建红1,3,袁燕1,3,卞建春1,3,刘学忠1,3*
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| (1.扬州大学 兽医学院/教育部农业与农产品安全国际合作联合实验室, 江苏 扬州 225009;2.扬州大学 化学化工学院, 江苏 扬州 225009;3.江苏高校动物重要疫病与人兽共患病防控协同创新中心, 江苏 扬州 225009) |
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| 摘要: |
| 为获得纯度更高的吡虫啉人工半抗原，通过对常规半抗原合成的反应顺序、试剂等进行调整和优化，筛选出最适3-巯基丙酸反应比例，采用薄层层析色谱法、质谱法和核磁共振法对合成的吡虫啉半抗原分子结构进行确证，进一步用合成的吡虫啉半抗原与载体蛋白偶联得到吡虫啉完全抗原并免疫小鼠，于三免和五免7天后断尾采血，采用间接ELISA法测定小鼠血清抗体效价。结果表明:1)吡虫啉、反应体系、有机相和水相的比移值分别为0.55、0.23、0.50和0；2)质荷比为326的峰是吡虫啉半抗原的分子离子峰，且其余杂质离子峰的数量明显下降；3)核磁共振结果与吡虫啉半抗原相符，3-巯基丙酸氧化副产物峰下降；4)三免后，小鼠血清效价达到1∶12 800，五免后，小鼠血清效价可达1∶51 200。综上，此优化方法不仅保证3-巯基丙酸完全反应，还减少3-巯基丙酸氧化副产物和不必要杂质的产生，提供了一种操作简单、安全，同时可减少半抗原合成过程杂质产生的吡虫啉半抗原合成优化方法，为后续吡虫啉单克隆抗体的制备提供便利。 |
| 关键词: 吡虫啉 半抗原 合成 优化 |
| DOI：10.11841/j.issn.1007-4333.2022.05.23 |
| 投稿时间：2021-07-11 |
| 基金项目:江苏省农业科技自主创新资金项目(CX(18)3022)；江苏省高校优势学科建设工程资助项目(PAPD) |
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| Optimization of artificial hapten synthesis of imidacloprid |
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ZHONG Yixuan1,3,WANG Yanhong1,3,YANG Chenggen1,2,ZOU Hui1,3,GU Jianhong1,3,YUAN Yan1,3,BIAN Jianchun1,3,LIU Xuezhong1,3*
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| (1.College of Veterinary Medicine/Joint Laboratory for International Cooperation on Agricultural and Agricultural Product Safety, Ministry of Education, Yangzhou University, Yangzhou 225009, China;2.College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou 225009, China;3.Collaborative Innovation Center for Prevention and Control of Important Animal and Human-Veterinary Diseases in Jiangsu Universities, Yangzhou 225009, China) |
| Abstract: |
| In order to obtain imidacloprid artificial semi-antigen with higher purity, the reaction sequence and reagents of conventional semi-antigen synthesis were adjusted and optimized, and the most suitable reaction ratio of 3-mercaptopropionic acid was selected. The molecular structure of imidacloprid haten was confirmed by thin layer chromatography, mass spectrometry and nuclear magnetic resonace(NMR). Mice were immunized with imidacloprid complete antigen by coupling the synthesized imidacloprid semi-antigen with carrier protein. The serum antibody titer of mice was determined by indirect ELISA after the third and the fifth immunization. The results showed that: 1)The specific shift values of imidacloprid, reaction system, organic phase and aqueous phase were 0. 55, 0. 23, 0. 50 and 0, respectively; 2)The peak of mass-to-charge ratio 326 was the molecular ion peak of imidacloprid half-antigen, and the number of the remaining impurity ion peaks decreased significantly; 3)The results of NMR analysis were consistent with imidacloprid half-antigen, and the oxidation by-product peak of 3-mercaptopropionic acid decreased; 4)After the third immunization, the serum titer of mice reached 1∶12 800, and the serum titer of mice reached 1∶51 200 after the fifth immunization. In conclusion, the improved method can not only ensure the complete reaction of 3-mercaptopropionic acid, but also reduce the generation of 3-mercaptopropionic acid oxidation by-products and unnecessary impurities. It provides a simple and safe method for the synthesis of imidacloprid semi-antigen, which can also reducee the impurities in the synthesis of semi-antigens. The approach could eventually facilitate the preparation of subsequent monoclonal antibodies to imidacloprid. |
| Key words: imidacloprid semi-antigen synthesis optimization |
