斑蝥素半抗原、人工抗原合成及多克隆抗体制备
摘要
斑蝥素(Cantharidin)存在于芫菁等昆虫体内,对多种害虫有良好的杀虫活性。本研究以斑蝥素为研究对象,从芫菁中提取斑蝥素并开展斑蝥素多克隆抗体制备研究,针对不同抗原-抗体反应特点建立了间接竞争ELISA方法。取得如下结果:
1.选择低毒的二氯甲烷为提取溶剂,在常规水解实验基础上,运用超声波萃取法对回收残渣进行提取工艺的改进,补充改进了斑蝥素提取工艺流程:超声波常温提取,萃取3次,料液比1:5(M/V)。通过改进的工艺,斑蝥素在回收残渣中依然提取出4.51g,表明通过常规水解法提取后,依然存在相当量斑蝥素存在残渣中。补充后的提取工艺简单易行,稳定性好。
2.斑蝥素没有直接与选用的载体蛋白偶联的基团,不能直接用于人工抗原的合成,必须加以改造形成含羧基的物质才能偶联,合成人工抗原以便产生抗体。本实验采用不同反应温度在两种不同溶剂中以斑蝥素、甘氨酸为原料,合成了一种半抗原--含羧基斑蝥素衍生物-氮羧甲基斑蝥素,通过对展开剂的选择,确定了最佳流动相V(氯仿):V(丙酮)=3:2,柱层析分离纯化,并利用IR、MS、1HNMR对其结构进行了表征。结果表明:该合成路线采用温度120℃、溶剂DMF为反应条件,产率达80%,且操作简便,重现性好。
3.利用碳二亚胺法(EDC),成功制备了稳定性好的免疫抗原Hapten-BSA和检测抗原Hapten-OVA。免疫抗原浓度为1.42 mg/mL,检测抗原浓度为2.27 mg/mL。200~400nm下紫外扫描证明合成成功。
4.斑蝥素免疫抗原Hapten-BSA免疫5只新西兰大白兔,采用间接ELISA法测定抗血清效价,结果表明:2#兔免疫效果最佳,获得针对斑蝥素的多克隆抗体效价达6.4×104。在此基础上通过试验确定了间接竞争ELISA方法检测斑蝥素的最佳工作浓度为:包被抗原稀释2000倍、抗体稀释4000倍。
这些结果为今后深入研究斑蝥素的细胞与分子毒理提供了物质基础。
Cantharidin is extracted from some insect species, especially the mylabris. It could be a good insecticide to a wide range of pests and a new type of biological pesticides. Based on the systematic study of cantharidin, the Entomology Museum of Northwest A&F University has developed several patent products with the major components of cantharidin. In this study, cantharidin is extracted from mylabris and used for polyclonal-antibody preparation, and an indirect competitive ELISA method is established. The results are as follows:
1.In this research, methylene dichloride solvent is selected as extraction. Based on the conventional hydrolyze experiments, ultrasonic extraction process is used to recycle residues: residues are extracted 3 times in room temperature, and the ratio of material to liquid is 1:5. This process is a supplement and improvement to cantharidin hydrolysis extraction. Through this improved technology, 4.51g cantharidin is extracted from recycled residues.This indicates that through conventional hydrolysis extraction, a considerabale amount of cantharidin still exists in the residues. The improved extracting method is stable and easy to operate.
2. Cantharidin can not be directly coupled with protein conjugates. It has to be modified so that it carries a carboxyl group. By adopting different temperatures in two different solvent, the research successfully systhesizes a cantharidin hapten with a carboxyl group. By studying on the developer, with V(chloroform):V(acetone)=3:2 as the best mobile Phase. Purified by silicagel column chromatography, the complex is evidenced by IR, NMR and MS. Reacting at 120℃and with DMF , the yield has reached 80%. This method is rapid and reproducible.
3.Using carbodiimide (EDC) method to sucessfully couple immune antigen Hapten-BSA and peridium antigen Hapten-OVA. The concentration of Hapten-BSA has reached 1.42 mg/mL, and the concentration of Hapten-BSA has reached 2.27 mg/mL . The 200~400nm UV scanning proves that the immune and peridium antigen required for peridium have been successfully synthesized.
4. After immunizing of five New Zealand rabbits with cantharidin immune antigen, indirect ELISA was used for the determination of the antibody titer. The high-titer cantharidin polyclonal-antibody is extracted from the 2# rabbit serumm and the highest titer was 6.4×104. Based on the experiment, the best concentration is: peridium antigen diluted for 2000-times and antibody diluted for 4000-times.
The research is just a preliminary study and further research in cantharidin cell and molecular toxicology is needed.
1.选择低毒的二氯甲烷为提取溶剂,在常规水解实验基础上,运用超声波萃取法对回收残渣进行提取工艺的改进,补充改进了斑蝥素提取工艺流程:超声波常温提取,萃取3次,料液比1:5(M/V)。通过改进的工艺,斑蝥素在回收残渣中依然提取出4.51g,表明通过常规水解法提取后,依然存在相当量斑蝥素存在残渣中。补充后的提取工艺简单易行,稳定性好。
2.斑蝥素没有直接与选用的载体蛋白偶联的基团,不能直接用于人工抗原的合成,必须加以改造形成含羧基的物质才能偶联,合成人工抗原以便产生抗体。本实验采用不同反应温度在两种不同溶剂中以斑蝥素、甘氨酸为原料,合成了一种半抗原--含羧基斑蝥素衍生物-氮羧甲基斑蝥素,通过对展开剂的选择,确定了最佳流动相V(氯仿):V(丙酮)=3:2,柱层析分离纯化,并利用IR、MS、1HNMR对其结构进行了表征。结果表明:该合成路线采用温度120℃、溶剂DMF为反应条件,产率达80%,且操作简便,重现性好。
3.利用碳二亚胺法(EDC),成功制备了稳定性好的免疫抗原Hapten-BSA和检测抗原Hapten-OVA。免疫抗原浓度为1.42 mg/mL,检测抗原浓度为2.27 mg/mL。200~400nm下紫外扫描证明合成成功。
4.斑蝥素免疫抗原Hapten-BSA免疫5只新西兰大白兔,采用间接ELISA法测定抗血清效价,结果表明:2#兔免疫效果最佳,获得针对斑蝥素的多克隆抗体效价达6.4×104。在此基础上通过试验确定了间接竞争ELISA方法检测斑蝥素的最佳工作浓度为:包被抗原稀释2000倍、抗体稀释4000倍。
这些结果为今后深入研究斑蝥素的细胞与分子毒理提供了物质基础。
Cantharidin is extracted from some insect species, especially the mylabris. It could be a good insecticide to a wide range of pests and a new type of biological pesticides. Based on the systematic study of cantharidin, the Entomology Museum of Northwest A&F University has developed several patent products with the major components of cantharidin. In this study, cantharidin is extracted from mylabris and used for polyclonal-antibody preparation, and an indirect competitive ELISA method is established. The results are as follows:
1.In this research, methylene dichloride solvent is selected as extraction. Based on the conventional hydrolyze experiments, ultrasonic extraction process is used to recycle residues: residues are extracted 3 times in room temperature, and the ratio of material to liquid is 1:5. This process is a supplement and improvement to cantharidin hydrolysis extraction. Through this improved technology, 4.51g cantharidin is extracted from recycled residues.This indicates that through conventional hydrolysis extraction, a considerabale amount of cantharidin still exists in the residues. The improved extracting method is stable and easy to operate.
2. Cantharidin can not be directly coupled with protein conjugates. It has to be modified so that it carries a carboxyl group. By adopting different temperatures in two different solvent, the research successfully systhesizes a cantharidin hapten with a carboxyl group. By studying on the developer, with V(chloroform):V(acetone)=3:2 as the best mobile Phase. Purified by silicagel column chromatography, the complex is evidenced by IR, NMR and MS. Reacting at 120℃and with DMF , the yield has reached 80%. This method is rapid and reproducible.
3.Using carbodiimide (EDC) method to sucessfully couple immune antigen Hapten-BSA and peridium antigen Hapten-OVA. The concentration of Hapten-BSA has reached 1.42 mg/mL, and the concentration of Hapten-BSA has reached 2.27 mg/mL . The 200~400nm UV scanning proves that the immune and peridium antigen required for peridium have been successfully synthesized.
4. After immunizing of five New Zealand rabbits with cantharidin immune antigen, indirect ELISA was used for the determination of the antibody titer. The high-titer cantharidin polyclonal-antibody is extracted from the 2# rabbit serumm and the highest titer was 6.4×104. Based on the experiment, the best concentration is: peridium antigen diluted for 2000-times and antibody diluted for 4000-times.
The research is just a preliminary study and further research in cantharidin cell and molecular toxicology is needed.
引文
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