CN106967140B - 一种多杀菌素半抗原及其制备方法和应用 - Google Patents
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Abstract
本发明公开了一种半抗原及其制备方法和应用,具体涉及一种多杀菌素半抗原。本发明还公开了所述多杀菌素半抗原的制备方法及其应用。本发明提供的多杀菌素半抗原既最大程度地保留了多杀菌素的化学结构,又通过化学合成改造引入了可以与蛋白质偶联的‑COOH,合成方法简单,产物纯度、得率较高;用该半抗原作为原料制备适于动物免疫的抗原体系,免疫动物后所得抗体效价、特异性、亲和力均较好,可用于制备酶联免疫试剂盒;该试剂盒操作简单、检测成本低、检测方法高效、准确、快速,可同时检测大批量的样本,适于动物源性食品中多杀菌素残留的现场监控和大量样本的筛查。本发明的多杀菌素半抗原在多杀菌素的检测中发挥重要作用。
Description
技术领域
本发明涉及一种半抗原及其制备方法和应用,尤其涉及一种多杀菌素半抗原及其制备方法和应用。
背景技术
多杀菌素(Spinosad)是从土壤放线菌刺糖多孢菌(Saccharopolyspora spinosa)分离出来的一种广谱杀虫剂,具有杀虫活性高,易降解、对有益生物毒性低等优点,在世界范围内被广泛应用于多种农作物害虫的防治,而多杀菌素残留超标可能会影响食品安全以及环境安全,因此迫切需要建立灵敏度高,特异性强,简便易行的多杀菌素检验方法。
多杀菌素检测方法包括高效液相色谱法、酶联免疫测定法。高效液相色谱法精确可靠、灵敏度高,检测极限可达到0.01μg/g,但前处理步骤多,回收率偏低。酶联免疫测定具有灵敏度高、特异性强、对仪器设备和人员素质要求低以及样品前处理简单等优点,适于现场监控和大规模样品筛选。
发明内容
本发明的目的是提供一种多杀菌素半抗原及其制备方法和应用。
本发明的第一个方面是提供一种多杀菌素半抗原,其分子结构式为:
本发明的第二个方面是提供一种如本发明第一个方面所述的多杀菌素半抗原的制备方法,包括以下步骤:步骤1,取多杀菌素,加入0.8-1.2N硫酸(H2SO4)中,75-85℃反应完全,纯化,得到C-17-多杀菌素,其分子结构式如式(2)所示,其中多杀菌素、硫酸的用量比为:1g︰10-20mL;步骤2,取C-17-多杀菌素,加入二氯甲烷溶解,加入丁二酸酐、4-二甲氨基吡啶,室温反应完全,纯化,得到权利要求1所述的多杀菌素半抗原,其中,C-17-多杀菌素、丁二酸酐和4-二甲氨基吡啶的用量比为:600mg︰100-750mg︰12.2-122mg,
优选地,上述步骤(1)中,硫酸的浓度为1N。
优选地,上述步骤(1)中,杀菌素、硫酸的用量比为:1g︰12-18mL,更优选为1g︰15mL。优选地,上述步骤(2)中,C-17-多杀菌素、丁二酸酐和4-二甲氨基吡啶的用量比为:600mg︰400-750mg︰80-122mg,更优选为:600mg︰750mg︰122mg。
本发明的第三个方面是提供一种多杀菌素抗原,由本发明第一个方面所述的多杀菌素半抗原与载体蛋白偶联得到,其分子结构式为:
本发明的第四个方面是提供一种制备如本发明第三个方面所述的多杀菌素抗原的方法,包括以下步骤:步骤a,取权利要求1所述的多杀菌素半抗原,加入无水二甲基甲酰胺(DMF)混匀后,加入二环己基碳二亚胺(DCC)和N-羟基琥珀酰亚胺(NHS),室温反应过夜,其中,多杀菌素半抗原、二环己基碳二亚胺和N-羟基琥珀酰亚胺的用量比为:0.09mmol︰0.09-0.18mmol︰0.09-0.18mmol;步骤b,离心,取溶液,将溶液缓慢加入载体蛋白溶液中,所用载体蛋白的量按照半抗原与载体蛋白的摩尔比15-40︰1计;步骤c,反应完全后,透析,得到多杀菌素抗原。
优选地,上述步骤a中,多杀菌素半抗原、二环己基碳二亚胺和N-羟基琥珀酰亚胺的用量比为:0.09mmol︰0.12mmol︰0.12mmol。
本发明的第五个方面是提供一种多杀菌素抗体,由本发明第三个方面所述的多杀菌素抗原免疫动物制备得到。
本发明的第六个方面是提供由本发明第五个方面所述的多杀菌素抗体制备的多杀菌素酶联免疫试剂盒。
本发明的第七个方面是提供本发明第一个方面所述的多杀菌素半抗原或本发明第三个方面所述的多杀菌素抗原在制备多杀菌素抗体中的应用。
本发明的第八个方面是提供本发明第五个方面所述的多杀菌素抗体或本发明第六个方面所述的多杀菌素酶联免疫试剂盒在检测动物源性食品中多杀菌素残留的应用。
本发明提供的多杀菌素半抗原既最大程度地保留了多杀菌素的化学结构,又通过化学合成改造引入了可以与蛋白质偶联的-COOH,合成方法简单,纯度、产率较高;用该半抗原作为原料,制备适于动物免疫的抗原体系免疫动物,所得抗体的效价、特异性、亲和力都比较好;所得的抗体用于酶联免疫试剂盒,使用方便、检测成本低、检测方法高效、准确、快速、可同时检测大批量的样本,适于动物源性食品中多杀菌素残留的现场监控和大量样本的筛查。本发明的多杀菌素半抗原在多杀菌素的检测中发挥重要作用。
附图说明
图1为多杀菌素半抗原合成路线图。
图2为多杀菌素半抗原核磁共振碳谱图。
图3为多杀菌素ELISA标准曲线。
具体实施方式
下面结合具体的实施例来进一步阐述本发明。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:多杀菌素半抗原的合成
步骤1:
称取1g即1.37mmol多杀菌素于50ml茄型烧瓶中,溶解于30ml 1N硫酸中,加热至80℃,磁力搅拌反应,TLC板监测反应完成(展开剂为乙酸乙酯︰石油醚=7︰3);用50ml稀硫酸洗涤沉淀,加入30ml二氯甲烷溶解,转移至分液漏斗,用饱和氯化钠溶液洗涤两次,收集有机相;用无水硫酸钠干燥后,过滤旋干溶剂,经200-300目硅胶柱层析纯化得到半抗原中间体,洗脱剂为乙酸乙酯︰石油醚,体积比为7︰3,得率为71.4%。
步骤2:
称取600mg半抗原中间体于棕色瓶中,加入750mg丁二酸酐与122.2mg 4-二甲氨基吡啶,溶于20ml二氯甲烷中,室温搅拌,TLC板监控至反应完全(展开剂为乙酸乙酯︰二氯甲烷︰醋酸=7︰3︰0.1);氮气吹干后,用50ml乙酸乙酯溶解沉淀,并用饱和氯化铵溶液洗涤两次,再用20ml乙酸乙酯反提水相,合并有机相,用无水硫酸钠干燥后,过滤旋干溶剂,经经200-300目硅胶柱层析纯化得到半抗原,洗脱剂为乙酸乙酯︰二氯甲烷︰醋酸,体积比为7︰3︰0.1,得率为80%。
实施例2:多杀菌素半抗原的鉴定
取上述实施例1的产物经核磁共振氢谱和碳谱测定,核磁共振数据如下,说明半抗原合成成功:
1H NMR(500MHz,CD3OD)δ7.09(s,1H),5.93(d,J=9.8Hz,1H),5.86(dt,J=9.8,2.8Hz,1H),5.03–4.95(m,1H),4.87(d,J=1.7Hz,2H),4.70–4.61(m,1H),4.34(dd,J=12.7,7.1Hz,1H),3.60–3.52(m,3H),3.52(s,3H),3.48–3.44(m,9H),3.43(t,J=3.3Hz,1H),3.35(s,6H),3.08(dt,J=18.8,7.2Hz,2H),2.98-2.96(m,1H),2.91–2.83(m,1H),2.64–2.58(m,4H),2.47(dd,J=13.4,3.2Hz,1H),2.41–2.30(m,1H),2.22–2.12(m,1H),2.04–1.93(m,1H),1.66–1.27(m,11H),1.24(d,J=6.2Hz,3H),1.09(d,J=6.7Hz,3H),0.99–0.88(m,1H),0.83(t,J=7.5Hz,3H).
13C NMR(126MHz,MeOD)δ203.44,175.96,173.93,173.92,150.37,145.06,130.66,129.74,97.06,83.55,82.52,78.63,77.96,77.91,76.29,69.10,61.14,59.15,57.72,51.06,49.85,47.46,46.72,42.87,42.55,38.54,37.44,35.01,33.58,31.11,30.27,29.84,29.28,22.34,18.13,16.45,9.62.
实施例3:多杀菌素抗原
向0.09mmol所述琥珀酰基多杀菌素半抗原中加入0.6ml的无水DMF,混匀后,加入0.12mmol的DCC和0.12mmol的NHS活化半抗原上的羧基基团,搅拌反应过夜后,离心去沉淀,将溶液平均分为三份,分别逐滴加入到不同的载体蛋白溶液中,所述载体蛋白为BSA,OVA,KLH等,所述溶解蛋白的缓冲液为碳酸盐缓冲液或硼酸盐缓冲液,所用蛋白的量按照半抗原与蛋白的摩尔比15-40︰1计算。待羧基活化的半抗原与蛋白分子上的氨基偶联反应过夜后,将反应液转移到半透膜中,使用10mM的pH7.2的PBS缓冲液于4℃透析3-5天,得到包被原,经紫外鉴定,多杀菌素半抗原与载体蛋白偶联成功。将透析完毕的溶液按照所用载体蛋白的量用PBS稀释成1mg/ml。
实施例4:抗血清制备
将1mg/ml的多杀菌素-KLH或多杀菌素-BSA作为免疫原与等体积的弗氏佐剂混合搅拌乳化,首次免疫用弗氏完全佐剂,以后免疫使用弗氏不完全佐剂,每间隔10至15天对小鼠免疫一次,第三次免疫后小鼠眼眶采血检测血清效价和抗体特异性。
实施例5:血清效价及抗体特异性检测
将1mg/ml的多杀菌素-OVA包被原使用碳酸盐缓冲液稀释合适的倍数后,加入到96孔酶标板中于37℃包被3小时,将多杀菌素标准品使用样品稀释液(PBS中含有0.1%的吐温20和明胶)稀释后,每孔加入50ul,以样品稀释液作为非抑制孔对照,随后加入经过样品稀释液稀释合适倍数的血清,于37℃温浴30min后,洗脱,再加入羊抗鼠酶标二抗反应30min,洗脱酶标二抗后,加入含有OPD或TMB的底物缓冲液显色5-10min,于酶标仪检测OD值。
本发明以多杀菌素-OVA作为包被原,多杀菌素-KLH作为免疫原所获得的抗血清,所建立的ELISA标准曲线如图3所示,抑制中浓度为287ng/ml。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (8)
1.一种多杀菌素半抗原,其特征在于,其分子结构式为:
2.一种如权利要求1所述的多杀菌素半抗原的制备方法,其特征在于,包括以下步骤:
步骤1,取多杀菌素,加入0.8-1.2N硫酸中,75-85℃反应完全,纯化,得到C-17-多杀菌素,其分子结构式如式(2)所示,其中多杀菌素、硫酸的用量比为:1g︰10-20mL;
步骤2,取C-17-多杀菌素,加入二氯甲烷溶解,加入丁二酸酐、4-二甲氨基吡啶,室温反应完全,纯化,得到权利要求1所述的多杀菌素半抗原,其中,C-17-多杀菌素、丁二酸酐和4-二甲氨基吡啶的用量比为:600mg︰100-750mg︰12.2-122mg,
3.一种多杀菌素抗原,其特征在于,由权利要求1所述的多杀菌素半抗原与载体蛋白偶联得到,其分子结构式为:
4.一种制备如权利要求3所述的多杀菌素抗原的方法,其特征在于,包括以下步骤:
步骤a,取权利要求1所述的多杀菌素半抗原,加入无水二甲基甲酰胺混匀后,加入二环己基碳二亚胺和N-羟基琥珀酰亚胺,室温反应过夜,其中,多杀菌素半抗原、二环己基碳二亚胺和N-羟基琥珀酰亚胺的用量比为:0.09mmol︰0.09-0.18mmol︰0.09-0.18mmol;
步骤b,离心,取溶液,将溶液缓慢加入载体蛋白溶液中,所用载体蛋白的量按照半抗原与载体蛋白的摩尔比15-40︰1计;
步骤c,反应完全后,透析,得到多杀菌素抗原。
5.一种多杀菌素抗体,其特征在于,由权利要求3所述的多杀菌素抗原免疫动物制备得到。
6.由权利要求5所述的多杀菌素抗体制备的多杀菌素酶联免疫试剂盒。
7.权利要求1所述的多杀菌素半抗原或权利要求3所述的多杀菌素抗原在制备多杀菌素抗体中的应用。
8.权利要求5所述的多杀菌素抗体或权利要求6所述的多杀菌素酶联免疫试剂盒在检测动物源性食品中多杀菌素残留的应用。
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