腐霉利人工抗原制备研究

doi:10.11924/j.issn.1000-6850.casb2021-0017

摘要/Abstract

摘要：

为了制备腐霉利(PCM)人工抗原,采用琥珀酸酐法改造PCM分子,利用碳二亚胺(EDC)法将改造后的PCM与牛血清白蛋白(BSA)及卵清蛋白(OVA)偶联制备PCM人工抗原PCM-BSA和PCM-OVA。采用凝胶电泳、紫外扫描及动物免疫法对人工抗原质量进行了鉴定。凝胶电泳及紫外扫描结果表明PCM与载体蛋白偶联成功;用PCM-BSA免疫小鼠的血清可以与PCM-OVA发生免疫反应,血清效价可以达到1:6400。用游离的PCM抑制酶标板上附着的PCM-OVA与小鼠血清反应时,即使游离PCM浓度达到256 µg/mL时,也没有出现明显的抑制;而用本研究制备的PCM-OVA溶液替代PCM时,则可以抑制酶标板上附着的PCM-OVA与小鼠血清之间的反应,且半数抑制浓度(IC₅₀)是在制备的PCM-OVA溶液稀释到320倍时。本研究将PCM成功地偶联到载体蛋白上,但免疫小鼠产生的针对PCM的抗体灵敏度比较低,这说明苯环可能是PCM分抗原决定簇的一部分,而琥珀酸酐改造PCM分子时破坏了抗原决定簇,因此该方法不适合用于PCM人工抗原制备。

关键词: 腐霉利, 分子改造, 人工抗原, 动物免疫, 血清

Abstract:

To prepare the artificial antigen of procymidone, the succinic anhydride method was applied to modify the structure of procymidone, and the modified procymidone was coupled with bovine serum albumin (BSA) and ovalbumin (OVA) by 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) method to synthetize PCM-BSA and PCM-OVA. SDS-PAGE, UV scanning and animal immunization were used to identify the quality of artificial antigen of PCM. The results of SDS-PAGE and UV scanning showed that PCM was coupled with the carrier proteins successfully. The serum of mice immunized by PCM-BSA could react with PCM-OVA; the serum titer could be 1:6400. The PCM was applied as an inhibitor to prevent the reaction between serum and PCM-OVA. However, even the concentration of PCM reached to 256 µg/mL, there was no significant inhibition. When PCM was replaced by the prepared PCM-OVA solution, which could inhibit the reaction between serum and PCM-OVA coated on the microtiter plates, the 50% inhibition concentration (IC₅₀) was 320 times diluted of the prepared PCM-OVA solution. In this study, PCM was coupled with carrier proteins successfully, but the serum sensitivity of mouse immunized with PCM artificial antigen was not high, which indicated that the benzene ring might be part of antigenic determinant of PCM. The epitope of PCM was destroyed during molecular modification by succinic anhydride, so this method was not suitable for the preparation of PCM artificial antigen.

Key words: procymidone, structural modification, artificial antigen, animal immunization, serum

中图分类号:

R155.5

胡骁飞, 邢云瑞, 孙亚宁, 庞杏豪, 王耀. 腐霉利人工抗原制备研究[J]. 中国农学通报, 2021, 37(33): 142-147.

Hu Xiaofei, Xing Yunrui, Sun Yaning, Pang Xinghao, Wang Yao. Research on Preparation of Artificial Antigen of Procymidone[J]. Chinese Agricultural Science Bulletin, 2021, 37(33): 142-147.

图/表 6

参考文献 27

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项目		血清稀释倍数							空白
-	小鼠序号	1:200	1:400	1:800	1:1600	1:3200	1:6400	1:12800	空白
OD₄₅₀值	1	2.005	1.336	0.821	0.599	0.375	0.209	0.143	0.079
	2	1.511	0.723	0.430	0.311	0.358	0.097	0.060	0.065
	3	0.808	0.361	0.207	0.140	0.103	0.073	0.076	0.068

项目		血清稀释倍数							空白
-	小鼠序号	1:200	1:400	1:800	1:1600	1:3200	1:6400	1:12800	空白
OD₄₅₀值	1	2.005	1.336	0.821	0.599	0.375	0.209	0.143	0.079
	2	1.511	0.723	0.430	0.311	0.358	0.097	0.060	0.065
	3	0.808	0.361	0.207	0.140	0.103	0.073	0.076	0.068

项目		PCM浓度/(µg/mL)								阳性	空白
-	序号	256	128	64	32	16	8	4	2	阳性	空白
OD₄₅₀值	1	0.714	0.759	1.003	0.911	0.850	0.926	0.755	0.781	0.792	0.087
	2	0.838	0.873	0.932	0.911	0.912	0.941	0.840	0.845	0.864	0.053
	3	0.546	0.693	0.799	0.827	0.601	0.666	0.656	0.606	0.544	0.064

项目		PCM浓度/(µg/mL)								阳性	空白
-	序号	256	128	64	32	16	8	4	2	阳性	空白
OD₄₅₀值	1	0.714	0.759	1.003	0.911	0.850	0.926	0.755	0.781	0.792	0.087
	2	0.838	0.873	0.932	0.911	0.912	0.941	0.840	0.845	0.864	0.053
	3	0.546	0.693	0.799	0.827	0.601	0.666	0.656	0.606	0.544	0.064

项目		PCM-OVA稀释倍数						阳性	空白
-	序号	10	20	40	80	160	320	阳性	空白
OD₄₅₀值	1	0.289	0.390	0.477	0.601	0.700	0.813	1.514	0.051
	2	0.163	0.151	0.235	0.245	0.282	0.341	0.533	0.058
	3	0.209	0.308	0.454	0.543	0.677	0.686	1.352	0.052