CN105399711B - 一种克百威羧基化半抗原的合成方法 - Google Patents
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Abstract
本发明“一种克百威羧基化半抗原的合成方法”,涉及生物化工技术。本发明是新型的克百威羧基化半抗原合成方法,合成步骤简洁有效,得率高,避免了传统三光气合成方法中大量使用光气和甲苯的危害性。以呋喃酚为起始原料,通过酯化和胺解反应,引入一条含4个碳和羧基端的手臂链,形成羧基化半抗原。采用活性酯法制备克百威人工抗原,紫外光谱扫描和免疫动物实验表明本发明制备的抗原良好的免疫原性。本发明的半抗原及人工抗原可完全用于克百威免疫分析中,满足国内对克百威残留的检测需要,应用前景广阔。
Description
技术领域
本发明涉及一种克百威羧基化半抗原的制备方法,属于生物化工技术领域。
背景技术
克百威(Carbofuran, CAR), 化学名为2,3-二氢-2,2-二甲基-7-苯并呋喃基-N-甲基氨基甲酸酯,商品名为呋喃丹,是广谱性杀虫、杀线虫剂,具有触杀和胃毒作用。克百威属于氨基甲酸酯类农药,与胆碱酯酶不可逆结合,使得乙酰胆碱不能分解而蓄积在体内,影响神经中枢传导,具有神经毒性。克百威在酸性土壤中不易降解残留期长,可以被植物根部吸收并输送到植物体的各器官,也可能通过径流渗透污染地下水源,给食品安全和环境带来潜在威胁。目前,虽然克百威已经禁用于蔬菜,但仍由不少违法使用现象,导致克百威在果蔬中时有检出。因此,在加强克百威使用监管力度的同时,加强克百威农药残留的检测十分必要。
目前克百威的检测多采用仪器分析法,需要昂贵的仪器设备和专业技术人员,对检材要求较高,样品前处理和提纯过程费时费力,不能满足现代检测对方便、快速、准确的要求。酶联免疫吸附法和胶体金试纸条法操作简便,对样本要求不高,可实现现场快速检测的要求。克百威本身不含有可偶联的功能基团,需要进行半抗原从头合成,目前国内外克百威半抗原合成中普遍采用传统的三光气合成工艺,由于大量光气和甲苯的使用,极易危害健康,造成环境污染。因此,本发明以呋喃酚为原料,设计一种新的克百威半抗原合成方法,并制备了完全抗原,为建立克百威免疫检测技术提供基础。
发明内容
本发明针对目前克百威半抗原三光气传统合成方法的危害性,提供了一种新的克百威羧基化半抗原合成方法,制备得到的半抗原和人工抗原可用于克百威免疫分析方法研究。
本发明的技术方案:以克百威的水解产物呋喃酚为原料,首先与二(对硝基苯)碳酸酯和4-氨基丁酸甲酯盐酸盐反应生成中间体,再和三氟醋酸反应,合成克百威羧基化半抗原;然后利用改良活性酯法将半抗原和载体蛋白上偶联,制备克百威人工抗原。其中半抗原合成路线为
。
工艺步骤如下。
1. 克百威半抗原的合成。
(1)将四口烧瓶置于磁力搅拌器上,配置温度计。取一定量的呋喃酚溶于50mL 二氯甲烷(DCM)中,冰浴下加入三乙胺(Et3N),搅拌10分钟后,将二(对硝基苯)碳酸酯溶于15mL二氯甲烷中,缓慢滴加到上述反应体系中,控制反应摩尔比为呋喃酚:三乙胺:二(对硝基苯)碳酸酯=1:1.1:1;逐渐升温至30℃,继续搅拌反应1小时,TLC板监控反应完成(展开剂:石油醚:乙酸乙酯=10:1)。
(2) 称取一定量经三乙胺碱化的4-氨基丁酸甲酯盐酸盐(与呋喃酚摩尔比为1:1),溶于10mL二氯甲烷中,在冰浴条件下加入到上述反应液中,将温度升到室温并继续搅拌过夜;依次用50mL水和50mL饱和食盐水各洗涤2次;用无水硫酸钠干燥后,过滤除去干燥剂,滤液经真空旋干后经200-300目硅胶柱层析纯化得到半抗原中间体,洗脱剂为石油醚:乙酸乙酯,体积比为10:1,收率为58%。
(3)将步骤2得到的半抗原中间体溶于15mL 1,4-二氧六环中,加入18mL三氟醋酸(TFA)和15mL水,60℃加热反应4小时后泠却至室温;用30mL乙酸乙酯萃取2次,合并有机相后依次用30mL水,饱和碳酸氢钠,水,饱和食盐水各洗涤2次;用无水硫酸钠干燥后,过滤除去干燥剂,滤液经真空旋干后经200-300目硅胶柱层析纯化得到半抗原,洗脱剂为石油醚:乙酸乙酯,体积比为5:1,收率为42%。
2. 克百威人工抗原的合成。
(1)取制备好的克百威半抗原、N-羟基琥珀酰亚胺(NHS)溶于1mL无水N,N-二甲基甲酰胺(DMF)中,待其完全溶解后再加入碳二亚胺(EDC),控制摩尔比为半抗原:NHS:DCC=1:1.5:1.5;室温下搅拌反应过夜;离心去沉淀,收集上清液,此为A液。
(2)称取一定量的牛血清白蛋白(BSA)溶解在5mL 磷酸盐缓冲液(PBS,0.01 mol/L,pH 7.4)中,控制摩尔比为半抗原:BSA=40-100:1(摩尔比),此为B液。
(3)在冰水浴中,将A液缓慢滴加到B液中,磁力搅拌下4℃反应过夜,即得到克百威人工抗原混合液。
(4)将混合液移入透析袋中,于PBS缓冲液中4℃搅拌透析3天,每天更换3次PBS缓冲液;透析结束后离心取上清,使用冻干法将其制成粉末,即得到克百威人工抗原。
3. 克百威人工抗原的鉴定。
分别对半抗原、载体蛋白和人工抗原溶液进行紫外扫描,根据载体蛋白在偶联前后紫外扫描光谱的变化情况来初步判断人工抗原偶联是否成功。然后,将所制备的人工抗原免疫新西兰大白兔,4免后采集抗血清进行检测,判断所制备的人工抗原是否具有免疫原性。
本发明的有益效果:本发明是新型的克百威羧基化半抗原合成方法,该反应合成步骤简洁有效,避免了传统三光气合成方法中大量使用光气和甲苯的危害性,以呋喃酚为起始原料,通过酯化和胺解反应,引入一条含4个碳和羧基端的手臂链,最大程度暴露克百威抗原决定簇,可完全用于免疫分析中,为以后人们的研究提供了方便的途径,可以满足国内对克百威残留的检测需要。
附图说明
图1克百威羧基化半抗原的核磁图。
图2克百威人工抗原紫外扫描图。
具体实施方式
为更好的理解本发明,下面结合具体的实施例来进一步阐明,应理解,这些实施例仅用于说明本发明,而不用来限制本发明的范围。
实施例1 克百威羧基化半抗原的制备。
(1) 将四口烧瓶置于磁力搅拌器上,配置温度计。取1.64g即10mmol呋喃酚溶于50mL 二氯甲烷中,冰浴下加入1.11g三乙胺,搅拌10分钟后,将3.04g二(对硝基苯)碳酸酯溶于15mL二氯甲烷中,缓慢滴加到上述反应体系中,逐渐升温至30℃,继续搅拌反应1小时,TLC板监控反应完成(展开剂:石油醚:乙酸乙酯=10:1)。
(2) 取1.697g经三乙胺碱化的4-氨基丁酸甲酯盐酸盐,溶于10mL二氯甲烷中,在冰浴条件下加入到上述反应液中,将温度升到室温并继续搅拌过夜;依次用50mL水和50mL饱和食盐水各洗涤2次;用无水硫酸钠干燥后,过滤除去干燥剂,滤液经真空旋干后经200-300目硅胶柱层析纯化得到半抗原中间体1.8g,洗脱剂为石油醚:乙酸乙酯,体积比为10:1,收率为58%。
(3)将步骤2得到的半抗原中间体1.8g溶于15mL 1,4-二氧六环中,加入18mL三氟醋酸和15mL水,60℃加热反应4小时后泠却至室温;用30mL乙酸乙酯萃取2次,合并有机相后依次用30mL水,饱和碳酸氢钠,水,饱和食盐水各洗涤2次;用无水硫酸钠干燥后,过滤除去干燥剂,滤液经真空旋干后经200-300目硅胶柱层析纯化得到半抗原0.73g,洗脱剂为石油醚:乙酸乙酯,体积比为5:1,收率为42%。1H NMR (500 MHz, CDCl3) δ6.97 (d, J = 7.0Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.78 (t, J = 7.0 Hz, 8.0 Hz, 1H), 5.24(br, 1H), 3.34 (m, 2H), 3.03 (s, 2H), 2.46 (t, J = 7.0 Hz, 2H), 1.91 (m, 2H),1.49 (s, 6H)。
实施例2 克百威人工抗原的制备。
(1)称取29.3mg克百威半抗原和17.3mg N-羟基琥珀酰亚胺溶于1mL无水DMF中,待其完全溶解后再加入23.3mg 碳二亚胺,室温下搅拌反应过夜;离心去沉淀,收集上清液,此为A液。
(2)称取载体蛋白BSA83mg溶解在5mL 0.01 mol/L PBS溶液中,为B液。
(3)在冰水浴中,将A液缓慢滴加到B液中,磁力搅拌下4℃反应过夜,即得到克百威人工抗原混合液。
(4)将混合液移入透析袋中,于PBS缓冲液中4℃搅拌透析3天,每天更换3次PBS缓冲液;透析结束后离心取上清,使用冻干法将其制成粉末,即得到克百威人工抗原。
实施例3 克百威抗血清的制备。
用实施例2 制备的人工抗原作为免疫原,与弗氏完全佐剂等量混合,乳化完全后选取2月龄的健康雌性新西兰大白兔于颈背部皮下多点注射,每间隔3周加强免疫一次。四免后第10天耳缘静脉采血,离心得到抗血清。
实验结果表明,本发明制备的克百威人工抗原免疫兔子得到的抗血清,四免效价可达81000,对克百威具有良好的选择性识别能力,抗体特性良好。
Claims (1)
1.一种克百威羧基化半抗原的合成方法,其特征是以呋喃酚为原料,通过酯化和胺解反应,最终合成克百威羧基化半抗原,具体合成路线如下:
包括以下步骤:
(1)取1.64g即10mmol呋喃酚溶于50mL二氯甲烷中,冰浴下加入1.11g即11mmol三乙胺,搅拌10分钟后,将3.04g即10mmol二(对硝基苯)碳酸酯溶于15mL二氯甲烷中,缓慢滴加到上述反应体系中,逐渐升温至30℃,继续搅拌反应1小时,TLC板监控反应完成,所用展开剂为石油醚:乙酸乙酯=10:1;
(2)称取一定量经三乙胺碱化的4-氨基丁酸甲酯盐酸盐,所述4-氨基丁酸甲酯盐酸盐的量与步骤(1)所述呋喃酚摩尔比为1:1,溶于10mL二氯甲烷中,在冰浴条件下加入到上述反应液中,将温度升到室温并继续搅拌过夜;依次用50mL水和50mL饱和食盐水各洗涤2次;用无水硫酸钠干燥后,过滤除去干燥剂,滤液经真空旋干后经200-300目硅胶柱层析纯化得到半抗原中间体1.8g,洗脱剂为石油醚:乙酸乙酯,体积比为10:1,收率为58%;
(3)将步骤(2)得到的半抗原中间体1.8g溶于15mL 1,4-二氧六环中,加入18mL三氟醋酸和15mL水,60℃加热反应4小时后泠却至室温;用30mL乙酸乙酯萃取2次,合并有机相后依次用30mL水,饱和碳酸氢钠,水,饱和食盐水各洗涤2次;用无水硫酸钠干燥后,过滤除去干燥剂,滤液经真空旋干后经200-300目硅胶柱层析纯化得到半抗原0.73g,洗脱剂为石油醚:乙酸乙酯,体积比为5:1,收率为42%。
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