CN108059622B - 一种基于三嗪环结构限定性n-(2-胍基-乙亚氨基)-吗啉抗原、抗体及应用 - Google Patents
一种基于三嗪环结构限定性n-(2-胍基-乙亚氨基)-吗啉抗原、抗体及应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于药物残留分析检测技术领域。更具体地,涉及一种基于三嗪环结构限定性N-(2-胍基-乙亚氨基)-吗啉半抗原、人工抗原、抗体及应用。
背景技术
N-(2-胍基-乙亚氨基)-吗啉盐酸盐(Moroxydine Hydrochloride,简称盐酸吗啉胍,俗称“病毒灵”),其核心有效成分为N-(2-胍基-乙亚氨基)-吗啉,分子式为C6H13N5O·HCl,分子量为207.66。由于N-(2-胍基-乙亚氨基)-吗啉能有效降低或抑制病毒的DNA聚合酶、RNA聚合酶的活性和蛋白质的合成等,其作为一种非核苷类广谱抗病毒药物,不仅广泛的应用于治疗流感、疱疹和水痘等病毒引起的疾病,在养殖业和种植业病毒性疾病防治方面应用也较多。
目前可以从烟草、糙米、番茄、蜂蜜和动物组织等中检测到N-(2-胍基-乙亚氨基)-吗啉的残留,另外,在环境土壤中的残留情况也不太乐观,这些情况不仅会导致耐药性病毒的增加,而且为人们的健康埋下了安全隐患。因此对动植物源性食品和环境中N-(2-胍基-乙亚氨基)-吗啉残留的检测具有重要意义。
目前,对于药品和食品中N-(2-胍基-乙亚氨基)-吗啉的传统的检测方法,大多依靠常规仪器分析方法,如高效液相色谱法、液相色谱-电喷雾串联质谱法等方法。这些方法存在周期长、专业性强,费用高等缺点,难以满足现场快速检测的需要,在应用上不能广泛推广。基于抗原-抗体特异性识别的酶联免疫分析方法(ELISA)具有快速、灵敏、准确等优点,特别适用于现场进行高通量检测。另外,ELISA因其能弥补仪器法的上述不足,已在食品安全检测中发挥了重要作用。
而ELISA法对于抗原、免疫原的选择要求较高,小分子化合物(MW≤1000Da)一般不具有免疫原性,不能直接免疫动物产生特异性抗体,但可与抗体产生反应,即具有反应原性。如果将小分子连接到具有免疫原性的大分子(通常为蛋白质)上,做成人工抗原,就可能使机体产生免疫应答,产生针对小分子结构的抗体。因为不是任何小分子与大分子载体连接都能产生特异性抗体,所以小分子半抗原结构的设计非常重要。建立酶联免疫分析方法的关键在于其半抗原和人工抗原的设计和合成。
发明内容
本发明要解决的技术问题是克服上述现有N-(2-胍基-乙亚氨基)-吗啉检测技术存在的存在周期长、专业性强、成本高,不适宜现场大批量快速检测,以及无法实现N-(2-胍基-乙亚氨基)-吗啉的直接快速检测等问题、缺陷和不足,提供一种基于三嗪环结构限定性N-(2-胍基-乙亚氨基)-吗啉半抗原,同时提供由所述半抗原偶联载体蛋白制备的人工抗原,以及免疫实验动物后使机体产生特异的针对N-(2-胍基-乙亚氨基)-吗啉的抗体;可用于N-(2-胍基-乙亚氨基)-吗啉的检测,检测效率高、操作简便。
本发明的目的是提供一种N-(2-胍基-乙亚氨基)-吗啉半抗原。
本发明另一目的是提供一种N-(2-胍基-乙亚氨基)-吗啉人工抗原。
本发明另一目的是提供一种N-(2-胍基-乙亚氨基)-吗啉抗体。
本发明上述目的通过以下技术方案实现:
一种N-(2-胍基-乙亚氨基)-吗啉半抗原,结构如式I所示:
其中R为-H和-CH3。
当R为-H时,所述的N-(2-胍基-乙亚氨基)-吗啉半抗原的结构如式Ia所示:
当R为-CH3时,所述的N-(2-胍基-乙亚氨基)-吗啉半抗原的结构如式Ib所示:
所述N-(2-胍基-乙亚氨基)-吗啉半抗原(Ia和Ib)的制备方法如下:
(1)N-(2-胍基-乙亚氨基)-吗啉半抗原Ia的制备方法,包括以下步骤:在碳酸钾存在下,先将N-(2-胍基-乙亚氨基)-吗啉盐酸盐解离出盐酸以制备N-(2-胍基-亚胺甲基)-吗啉a,再与甲醇钠/甲酸乙酯反应得到N-(2-胍基-乙亚氨基)-吗啉半抗原Ia,其反应式为:
(2)N-(2-胍基-乙亚氨基)-吗啉半抗原Ib的制备方法,包括以下步骤:在甲醇钠存在下,先将N-(2-胍基-乙亚氨基)-吗啉盐酸盐解离出盐酸以制备N-(2-胍基-亚胺甲基)-吗啉a,再与乙酸乙酯反应得N-(2-胍基-乙亚氨基)-吗啉半抗原Ib,其反应式为:
在上述半抗原Ia N-(2-胍基-乙亚氨基)-吗啉半抗原Ia的制备方法中的关键因素:
(1)碳酸钾与N-(2-胍基-乙亚氨基)-吗啉盐酸盐的摩尔比为:1:1.2,溶剂蒸馏水,室温搅拌,产物需经减压旋蒸除去水相,再经甲醇溶解、过滤以除去杂质氯化钾和多余的碳酸钾,最终蒸干滤液可得N-(2-胍基-亚胺甲基)-吗啉a;
(2)所述的N-(2-胍基-亚胺甲基)-吗啉a、甲醇钠、甲酸乙酯的摩尔比为1:1.5:3,溶剂THF,60℃搅拌反应36h,期间用TLC监控,产物需柱层析分离提纯。
在上述N-(2-胍基-乙亚氨基)-吗啉半抗原Ib的制备方法中的关键因素:
(1)甲醇钠和N-(2-胍基-乙亚氨基)-吗啉盐酸盐的摩尔比为1:0.6~0.7,溶剂THF,室温搅拌,得到粗产物N-(2-胍基-亚胺甲基)-吗啉a,产物不经处理直接进入下一步反应;
(2)乙酸乙酯与上步中甲醇钠的摩尔比为2:1,乙酸乙酯与适量无水THF混合后边搅拌边滴加到粗产物N-(2-胍基-亚胺甲基)-吗啉a中,滴加完毕后,冷凝回流,期间用TLC监控,产物需柱层析分离提纯。
另外,所述N-(2-胍基-乙亚氨基)-吗啉半抗原在制备N-(2-胍基-乙亚氨基)-吗啉人工抗原方面的应用,也在本发明的保护范围之内。
一种N-(2-胍基-乙亚氨基)-吗啉人工抗原,是由所述N-(2-胍基-乙亚氨基)-吗啉半抗原与载体蛋白偶联得到。
所述N-(2-胍基-乙亚氨基)-吗啉人工抗原的结构式如式Ⅱ结构:
具体人工抗原具有式Ⅱa和式Ⅱb结构:
优选地,所述载体蛋白为牛血清蛋白(BSA)或卵清蛋白(OVA),其中,BSA用于制备免疫原,OVA用于制备包被原。
更优选地,所述N-(2-胍基-乙亚氨基)-吗啉人工抗原采用采用戊二醛法制备,具体步骤包括:将权利要求1所述N-(2-胍基-乙亚氨基)-吗啉半抗原溶于PBS缓冲溶液,与含有10mg/mL载体蛋白的PBS缓冲溶液混合,搅拌过程中逐滴加入适量的2.5%的戊二醛水溶液,室温避光反应3h,4℃用PBS透析2天,每天更换透析液3次,得到N-(2-胍基-乙亚氨基)-吗啉人工抗原,分装,并低温保存备用;所述的N-(2-胍基-乙亚氨基)-吗啉半抗原与载体蛋白的的摩尔比为40-100:1。
更优选地,所述N-(2-胍基-乙亚氨基)-吗啉人工抗原的制备方法为:将N-(2-胍基-乙亚氨基)-吗啉半抗原溶于PBS缓冲溶液(0.01M,pH 7.4)中,与含有10mg/mL载体蛋白的PBS缓冲溶液混合,搅拌过程中逐滴加入适量的2.5%的戊二醛水溶液,室温避光反应3h,4℃用PBS透析2天,每天更换透析液3次,得到N-(2-胍基-乙亚氨基)-吗啉人工抗原,分装,并低温保存备用;所述的N-(2-胍基-乙亚氨基)-吗啉半抗原与载体蛋白的的摩尔比为60:1。
另外,所述N-(2-胍基-乙亚氨基)-吗啉人工抗原在制备抗N-(2-胍基-乙亚氨基)-吗啉多克隆抗体、单克隆抗体或基因工程抗体方面的应用,也在本发明的保护范围之内。
一种N-(2-胍基-乙亚氨基)-吗啉抗体,以所述N-(2-胍基-乙亚氨基)-吗啉人工抗原免疫动物制备得到。所述的N-(2-胍基-乙亚氨基)-吗啉抗体为多克隆抗体、单克隆抗体或者基因工程抗体。
所述N-(2-胍基-乙亚氨基)-吗啉抗体在免疫法检测N-(2-胍基-乙亚氨基)-吗啉中的应用,也在本发明的保护范围之内。
一种直接检测N-(2-胍基-乙亚氨基)-吗啉的快速免疫分析方法,包括如下步骤:
(1)将所述的N-(2-胍基-乙亚氨基)-吗啉人工抗原作为免疫原免疫动物制备抗N-(2-胍基-乙亚氨基)-吗啉多克隆抗体;
(2)将所述的N-(2-胍基-乙亚氨基)-吗啉人工抗原作为包被原包被在微孔板上,将上步制备得到的抗N-(2-胍基-乙亚氨基)-吗啉多克隆抗体加入微孔板中;
(3)采用间接竞争ELISA方法检测抗血清效价。
本发明具有以下有益效果:
本发明提供了一种N-(2-胍基-乙亚氨基)-吗啉半抗原,以及基于此半抗原制备的人工抗原,阐述了两者的制备方法以及在抗体制备方面的应用。
本发明的抗原抗体合成过程简单、成本较低,并为开发出成本低廉,检测效率高、操作简便的酶联免疫快速检测工具,奠定了基础,具有良好的应用前景。
附图说明
图1为N-(2-胍基-乙亚氨基)-吗啉半抗原Ia(A)和N-(2-胍基-乙亚氨基)-吗啉半抗原Ib(B)的合成路线。
图2中A为本发明制备的N-(2-胍基-乙亚氨基)-吗啉半抗原Ia的质谱图;B为N-(2-胍基-乙亚氨基)-吗啉半抗原Ia的抗体性能鉴定图。
图3中A为本发明制备的N-(2-胍基-乙亚氨基)-吗啉半抗原Ib的质谱图;B为N-(2-胍基-乙亚氨基)-吗啉半抗原Ib的抗体性能鉴定图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1:N-(2-胍基-乙亚氨基)-吗啉半抗原Ia的合成
合成路线如图1所示,在25mL单口圆底烧瓶中,依次加入2.1g(10mM)N-(2-胍基-乙亚氨基)-吗啉盐酸盐、10mL蒸馏水,搅拌使固体溶解,加入1.6g(12mM)碳酸钾,室温下磁力搅拌30min,减压蒸干水相,得到白色固体粉末,用甲醇溶解该固体粉末,过滤除去生成的氯化钾和多余的碳酸钾,蒸干滤液得到N-(2-胍基-乙亚氨基)-吗啉a。然后在25ml单口圆底烧瓶中,依次加入342.4mg(2mM)N-(2-胍基-乙亚氨基)-吗啉a,162.1mg(3mM)甲醇钠和444.5mg(6mM)甲酸乙酯,再加入12ml THF,60℃搅拌反应36h,期间用TLC监控。蒸干溶剂,粗产品经柱层析(MeOH:TCM=1:10)分离提纯,得半抗原Ia。
实施例2:N-(2-胍基-乙亚氨基)-吗啉半抗原Ib的合成
合成路线如图1所示,将0.4g(7.0mM)甲醇钠与1.0g(4.8mM)N-(2-胍基-乙亚氨基)-吗啉盐酸盐置于50mL单口圆底烧瓶中,加入适量无水THF室温下搅拌。边搅拌边缓慢滴加1.2g(14.0mM)乙酸乙酯和10ml无水THF的混合溶液,滴加完毕后,80℃条件下冷凝回流36h,期间用TLC监控。反应结束后,蒸干溶剂,粗产品经柱层析(MeOH:TCM=1:10)分离提纯,得半抗原Ib。
实施例3:人工抗原的合成
本发明提供N-(2-胍基-乙亚氨基)-吗啉人工抗原的制备方法,主要包括免疫原和包被原的合成。免疫原与包被原的制备不同之处在于载体蛋白种类,所述的免疫原采用的载体蛋白为牛血清蛋白(BSA);所述的包被原采用的载体蛋白为卵清蛋白(OVA)。免疫原的制备方法。本发明采用的合成免疫原/包被原的方法为戊二醛法。
(1)免疫原的合成
将实施例1所述的N-(2-胍基-乙亚氨基)-吗啉半抗原Ia1.5mg(0.008mmol),溶于1mL的PBS缓冲溶液(0.01M,pH 7.4)中,与1mL 10mg/mL载体蛋白的PBS缓冲溶液混合,搅拌过程中逐滴加入100μL的2.5%的戊二醛水溶液,室温避光反应3h,4℃用PBS透析2天,每天更换透析液3次,得到N-(2-胍基-乙亚氨基)-吗啉免疫原Ia-BSA,用PBS调整浓度为1mg/mL,每管500μL分装于0.5mL离心管中。冻存于-20℃冰箱中,备用。
将上一步中的半抗原换成实施例2所述的N-(2-胍基-乙亚氨基)-吗啉半抗原Ib1.56mg,其它合成步骤相同,可得N-(2-胍基-乙亚氨基)-吗啉免疫原Ib-BSA,用PBS调整浓度为1mg/mL,每管500μL分装于0.5mL离心管中。冻存于-20℃冰箱中,备用。
(2)包被原的合成
对于权利要求1中所述的半抗原对应的包被原的合成方法,其合成步骤与免疫原的合成步骤一致,唯一不同的是将所用的载体蛋白换成OVA,由此可得包被原Ia-OVA、Ib-OVA。用PBS调整浓度为1mg/mL,每管500μL分装于0.5mL离心管中。冻存于-20℃冰箱中,备用。
实施例4:N-(2-胍基-乙亚氨基)-吗啉单克隆抗体、基因工程抗体和多克隆抗体的制备
1、单克隆抗体的制备
动物免疫:将实施例3制备的免疫原Ia-BSA、免疫原Ib-BSA与等量的佐剂(第一次用完全弗氏佐剂,之后均用不完全弗氏佐剂)混合乳化,对Balb/c小鼠进行间隔免疫,间接免疫分析检测并得到血液中含有N-(2-胍基-乙亚氨基)-吗啉特异性抗体的小鼠脾脏。
细胞融合与克隆:取产生特异性抗体的Balb/c小鼠脾细胞与骨髓瘤细胞SP20融合,采用间接竞争酶联免疫分析方法测定细胞上清液,筛选阳性孔。利用有限稀释法或显微克隆法对阳性孔进行克隆化,得到并建立产单克隆抗体的杂交瘤细胞株。
细胞冻存与复苏:取处于对数生长期的杂交瘤细胞用冻存液制成细胞悬液,分装于冻存管,在液氮中长期保存。复苏时取出冻存管,立即放入37℃水浴中速融,离心去除冻存液后,移入培养瓶内培养。
单克隆抗体的制备与纯化:采用体内诱生法,将Balb/c小鼠(8周龄)腹腔注入灭菌石蜡油,7-14天后腹腔注射杂交瘤细胞,7-10天后采集腹水。经辛酸-饱和硫酸胺法或亲和层析法进行腹水纯化,纯度经SDS-PAGE电泳鉴定,小瓶分装,-20℃保存。
2、基因工程抗体的制备
提取N-(2-胍基-乙亚氨基)-吗啉单克隆细胞或经N-(2-胍基-乙亚氨基)-吗啉免疫原免疫后的小鼠脾脏细胞的RNA,反转录为cDNA,设计抗体轻重链扩增引物,利用PCR技术扩增出抗体的轻重链基因,插入表达质粒TGI菌株,在大肠杆菌中表达,利用免疫亲和方法进行纯化得到基因工程抗体,纯度由SDS-PAGE电泳鉴定,小瓶分装,-20℃保存。
3、多克隆抗体的制备
将实施例3制备的免疫原Ia-BSA、免疫原Ib-BSA与等量的佐剂(第一次用完全弗氏佐剂,之后均用不完全弗氏佐剂)混合乳化,采用背部皮下、各部位皮下、腿部肌肉和耳缘静脉多种注射方式免疫体重为2.5~3kg的健康新西兰大白兔,每种免疫原对应注射两只。第一次免疫间隔四周后每三周加强免疫一次。第三次加强免疫后1周耳缘静脉取血,并利用间接ELISA测定血清效价,当效价不在上升时,采用耳缘静脉加强免疫。两天后心脏采血,室温静置0.5-1h,于4℃,12000r/min下离心10min,取上清分装于离心管中,于-20℃下保存备用。
4、抗体测试
图2中A为本发明制备的N-(2-胍基-乙亚氨基)-吗啉半抗原Ia的质谱图;B为N-(2-胍基-乙亚氨基)-吗啉半抗原Ia的抗体性能鉴定图。
图3中A为本发明制备的N-(2-胍基-乙亚氨基)-吗啉半抗原Ib的质谱图;B为N-(2-胍基-乙亚氨基)-吗啉半抗原Ib的抗体性能鉴定图。
间接竞争ELISA测定抗体效价以吸光值在1.0-1.5之间为准,结果表明,式Ia所对应的N-(2-胍基-乙亚氨基)-吗啉半抗原Ia对应的多克隆抗血清的效价是1:64000,抑制率81%;式Ib所对应的N-(2-胍基-乙亚氨基)-吗啉半抗原Ib对应的多克隆抗血清的效价是1:64000,抑制率81%。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (8)
2.一种N-(2-胍基-乙亚氨基)-吗啉人工抗原,其特征在于,是由权利要求1所述N-(2-胍基-乙亚氨基)-吗啉半抗原与载体蛋白偶联得到。
3.根据权利要求2所述N-(2-胍基-乙亚氨基)-吗啉人工抗原,其特征在于,所述载体蛋白为牛血清蛋白或卵清蛋白。
4.根据权利要求2所述N-(2-胍基-乙亚氨基)-吗啉人工抗原,其特征在于,采用戊二醛法制备,具体步骤包括:将权利要求1所述N-(2-胍基-乙亚氨基)-吗啉半抗原溶于PBS缓冲溶液,与含有10mg/mL载体蛋白的PBS缓冲溶液混合,搅拌过程中逐滴加入适量的2.5%的戊二醛水溶液,室温避光反应3h,4℃用PBS透析2天,每天更换透析液3次,得到N-(2-胍基-乙亚氨基)-吗啉人工抗原,分装,并低温保存备用;所述的N-(2-胍基-乙亚氨基)-吗啉半抗原与载体蛋白的的摩尔比为40-100:1。
5.权利要求2所述N-(2-胍基-乙亚氨基)-吗啉人工抗原在制备抗N-(2-胍基-乙亚氨基)-吗啉多克隆抗体、单克隆抗体或基因工程抗体方面的应用。
6.一种N-(2-胍基-乙亚氨基)-吗啉抗体,其特征在于,以权利要求2所述N-(2-胍基-乙亚氨基)-吗啉人工抗原制备得到。
7.权利要求6所述N-(2-胍基-乙亚氨基)-吗啉抗体在免疫法检测N-(2-胍基-乙亚氨基)-吗啉中的应用。
8.一种直接检测N-(2-胍基-乙亚氨基)-吗啉的快速免疫分析方法,其特征在于,包括如下步骤:
(1)将权利要求2所述N-(2-胍基-乙亚氨基)-吗啉人工抗原作为免疫原免疫动物制备抗N-(2-胍基-乙亚氨基)-吗啉多克隆抗体;
(2)将权利要求2所述N-(2-胍基-乙亚氨基)-吗啉人工抗原作为包被原包被在微孔板上,将上步制备得到的抗N-(2-胍基-乙亚氨基)-吗啉多克隆抗体加入微孔板中;
(3)采用间接竞争ELISA方法检测抗血清效价。
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