CN104341432A - 一种黄曲霉毒素半抗原、人工抗原及其制备方法 - Google Patents
一种黄曲霉毒素半抗原、人工抗原及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种黄曲霉毒素半抗原、人工抗原及其制备方法,将甲醇:双蒸水:吡啶溶液混合,将黄曲霉毒素与羧甲基羟胺半盐酸盐溶解于上述混合溶液中,对所得混合物85℃加热回流3~5h,直到所有的黄曲霉毒素完全转化为黄曲霉毒素半抗原。一种黄曲霉毒素人工抗原,采用EDC活性酯法合成。本发明制备的黄曲霉毒素人工抗原具有较好的免疫原性,可刺激机体产生效价较高的抗体,对于采用免疫分析技术快速检测残留的黄曲霉毒素B1具有重要意义;并且确立了一种非动物源的载体-大豆11S球蛋白酸性亚基作为新型的免疫原载体,拟代替牛血清白蛋白(BSA)。
Description
技术领域
本发明涉及一种食品安全领域,尤其涉及的是一种黄曲霉毒素半抗原、人工抗原及其制备方法。
背景技术
黄曲毒素(aflatoxin),也称作黄曲霉素,是一种有强烈生物毒性的化合物,常由黄曲霉及另外几种霉菌在霉变的谷物中产生,如大米、豆类、花生等,是目前为止最强的致癌物质。加热至280℃以上才开始分解,所以一般的加热不易破坏其结构。黄曲毒素主要有B1、B2、G1与G2等4种,又以B1的毒性最强。食米储存不当,极容易发霉变黄,产生黄曲毒素。
黄曲毒素与肝癌有密切关系,还会引起组织失血、厌食等症状。黄曲霉毒素中毒(Aflatoxicosis)主要对动物肝脏的伤害,受伤害的个体因动物种类年龄,性别和营养状态而异。研究结果表明黄曲霉毒素可导致肝功能下降,降低牛奶产量和产蛋率。并使动物的免疫力降低易受有害微生物的感染。此外,长期食用含低浓度黄曲霉毒素的饲料也可导致胚胎内中毒。通常年幼的动物对黄曲霉毒素更敏感。黄曲霉毒素的临床表现为消化系统功能紊乱降低生育能力。降低饲料利用率,贫血等。黄曲霉毒素不仅能够使奶牛的产奶量下降而且还使牛奶中含有转型的黄曲霉毒素M1和M2。据美国农业经济学家统计,由于食用黄曲霉毒素污染的饲料每年至少要使美国畜牧业遭受10%的经济损失。在中国,由此而带来的畜牧业损失可能会更大。黄曲霉毒素Bl的半数致死量为0.36毫克/公斤体重,属特剧毒的毒物范围(动物半数致死量<10毫克/公斤=它的毒性比氰化钾大10倍比砒霜大68倍)。它引起人的中毒主要是损害肝脏,发生肝炎肝硬化,肝坏死等。黄曲霉毒素是目前发现的最强的致癌物质。其致癌力是奶油黄的900倍比二甲基亚硝胺诱发肝癌的能力大75倍,比3,4苯并芘大4000倍。它主要诱使动物发生肝癌也能诱发胃癌,肾癌直肠癌及乳腺,卵巢小肠等部位的癌症。
因此黄曲霉毒素的检测是农产品质量安全检测的一个重要指标。黄曲霉毒素等真菌毒素可以通过酶联免疫分析的方法进行检测,而目前免疫检测中所用的抗原载体大多是动物源的载体,不仅不够安全,而且存在一定的免疫缺陷,如敏感性较差,特异性不够高等问题。
发明内容
本发明的目的在于克服现有技术的不足,提供了一种黄曲霉毒素半抗原、人工抗原及其制备方法。
本发明是通过以下技术方案实现的,一种黄曲霉毒素半抗原AFB1-O-CMO,分子结构式为:
一种黄曲霉毒素半抗原AFB1-O-CMO的制备方法,包括以下步骤:
(1)甲醇:双蒸水:吡啶溶液按体积比为4:1:1的比例混合后,再将黄曲霉毒素与半羧基羟胺盐酸CMO按质量比1:2混合后溶解于上述混合溶液中;
(2)对步骤(1)所得混合物80~90℃加热回流3~4h,直到所有的黄曲霉毒素完全转化为黄曲霉毒素半抗原;
(3)用旋转蒸发仪将溶剂旋干,加水后,用2mol/L的H2SO4调节PH=4,然后用5ml乙酸乙酯萃取2~3次,合并有机相,并用水洗涤至少2次,除去CMO,浓缩旋干液体,得到黄色油状半固态物质,即为黄曲霉毒素半抗原AFB1-O-CMO。
一种黄曲霉毒素人工抗原,所述人工抗原的分子结构式为:
所述载体蛋白Protein是大豆11S球蛋白酸性亚基。
一种黄曲霉毒素人工抗原的制备方法,采用EDC活性酯法合成,具体步骤如下:
(1)将黄曲霉毒素半抗原溶于2ml体积浓度为25%乙醇溶液中,称取4.5mg大豆11S球蛋白酸性亚基,溶于2ml纯水中,黄曲霉毒素半抗原与大豆11S球蛋白酸性亚基的投料摩尔比为60:1;
(2)加入50mg的EDC,室温下,避光搅拌反应48h;
(3)反应期间,再加入EDC各2次,每次50mg;
(4)反应产物在4℃搅拌下用PBS透析3天,每天换液3~6次,透析后得到纯化的AFB1-O-AS储存于-20℃。
所述大豆11S球蛋白酸性亚基的制备方法包括以下步骤:
(1)以1:5的体积比将大豆11S球蛋白溶于Tris-HCl缓冲液,加β巯基乙醇之后调节混合溶液的整体浓度至0.015mol.L-1,调pH为8.0,在90℃条件下水浴30min;
(2)4℃,10000r·min-1条件下离心20min,得到沉淀,调pH至5.0,冷冻干燥后即得大豆球蛋白碱性亚基;
(3)收集除去碱性亚基的上清液,调pH至5.0,4℃、6500r·min-1条件下离心20min分离沉淀,调pH至中性,冷冻干燥后即得大豆11S球蛋白酸性亚基。
一种黄曲霉毒素人工抗原用于制备黄曲霉毒素抗体。
将黄曲霉毒素人工抗原作为免疫抗原免疫雌性小鼠得到黄曲霉毒素多克隆抗体。
一种黄曲霉毒素抗体在检测黄曲霉毒素中的应用。
本发明针对动物源载体的生物安全性隐患及其在免疫应用上的不足之处,选取一种新型的非动物源的免疫载体-大豆11S球蛋白酸性亚基,同黄曲霉毒素小分子偶联建立黄曲霉毒素半抗原。大豆11S球蛋白酸性亚基结构稳定、溶解性强、在有机溶剂状态下都能和半抗原进行交联,而且同免疫动物的亲缘关系也比较远,一方面提高了黄曲霉毒素检测的灵敏度和特异性,也促进了免疫载体的生物安全性能的研究,为植物源蛋白在免疫生物学上的应用奠定理论基础,为蛋白质载体领域的进一步拓展提供了新的思路和可借鉴的实例。
本发明相比现有技术具有以下优点:本发明制备的黄曲霉毒素人工抗原具有较好的免疫原性,可刺激机体产生效价较高的抗体,对于采用免疫分析技术快速检测残留的黄曲霉毒素B1具有重要意义;并且确立了一种非动物源的载体-大豆11S球蛋白酸性亚基作为新型的免疫原载体,拟代替牛血清白蛋白(BSA)。
附图说明
图1是薄层层析法TLC鉴定示意图;
图2是AFB1和AFB1-O-CMO的紫外扫描光谱图;
图3是AFB1-O-CMO的质谱图;
图4是AFB1-O-AS和AFB1的紫外吸收光谱;
图5是AFB1-O-AS和AS的荧光发射光谱。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
1、黄曲霉毒素半抗原的制备
本实施例的一种黄曲霉毒素半抗原,分子结构式为
一种黄曲霉毒素半抗原的制备方法,包括以下步骤:
(1)甲醇:双蒸水:吡啶溶液按体积比为4:1:1的比例混合后,再将黄曲霉毒素与半羧基羟胺盐酸CMO按质量比1:2混合后溶解于上述混合溶液中;
(2)对上述混合物85℃加热回流3~4h,直到所有的黄曲霉毒素完全转化为黄曲霉毒素半抗原;肟化率达到98.2%,相对来说是比较高的,反应也较为彻底。
(3)用旋转蒸发仪将溶剂旋干,或剩余0.5ml,加水后,用2mol/L的H2SO4调节PH=4,然后用5ml乙酸乙酯萃取2~3次,合并有机相,并用水洗涤至少2次,除去CMO,浓缩旋干液体,得到黄色油状半固态物质,即为黄曲霉毒素半抗原AFB1-O-CMO。
黄曲霉毒素半抗原(AFB1-O-CMO)的鉴定:
(1)TLC鉴定
将肟化物以体积比9∶1的三氯甲烷和丙酮混合溶液为展开剂进行薄层层析鉴定。用毛细管在硅胶板上点样,放入层析缸中,待溶剂达到离硅胶板前沿1cm左后时,取出硅胶板放入硫酸乙醇显色剂中,进行显色。
如图1所示,Rf=0.25左右为黄曲霉毒素半抗原(AFB1-O-CMO),Rf=0.75左右的组分为未转化的AFB1,可知黄曲霉毒素半抗原占绝大部分,转化率极高。
(2)紫外扫描鉴定
将肟化物用少量的甲醇溶解,同时用甲醇配制浓度为0.03mg/mL的AFB1溶液,用紫外-可见光分光光度计分别进行200~400nm扫描。
如图2所示,在甲醇溶液中,AFB1-O-CMO有209,220,264,330nm4个吸收峰,和AFB1相比,两者吸收峰基本一致。
(3)电喷雾质谱鉴定
AFB1的相对分子质量为312.3,如图3所示,与此对应图中m/z408.06是AFB1羧甲基活化物的准分子离子峰[AFB1-O+Na]+,即活化物的相对分子质量为385.06,与应得产物C19H15NO8的相对分子质量基本一致。而图中m/z422.06可能是AFB1-O钠盐的氮偶合物的分子离子峰[AFB1-O-N+Na]+,图中m/z312.1是AFB1的准分子离子峰。综合UV结果,可初步断定活化产物是AFB1羧基活化物,半抗原合成成功。
2、黄曲霉毒素人工抗原的制备
一种黄曲霉毒素人工抗原,分子结构式为
所述载体蛋白Protein是大豆11S球蛋白酸性亚基。
一种黄曲霉毒素人工抗原的制备方法,采用EDC活性酯法合成,具体方法如下:
(1)将黄曲霉毒素半抗原溶于2ml体积浓度为25%乙醇溶液中,称取4.5mg大豆11S球蛋白酸性亚基,溶于2ml纯水中,黄曲霉毒素半抗原与大豆11S球蛋白酸性亚基的投料摩尔比为60:1;
(2)加入50mg的EDC,室温下,避光搅拌反应48h;
(3)反应期间,再加入EDC各2次,每次50mg;
(4)反应产物在4℃搅拌下用PBS透析3天,每天换液3~6次,透析后得到纯化的黄曲霉毒素人工抗原AFB1-O-AS储存于-20℃。
所述大豆11S球蛋白酸性亚基的制备方法包括以下步骤:
(1)将大豆11S球蛋白溶于Tris-HCl缓冲液(1:5),加β巯基乙醇调节混合溶液的整体浓度至0.015mol.L-1,调pH为8.0,在90℃条件下水浴30min;
(2)4℃,10000r·min-1条件下离心20min,得到沉淀,调pH至5.0,冷冻干燥后即得大豆球蛋白碱性亚基;
(3)收集除去碱性亚基的上清液,调pH至5.0,4℃、6500r·min-1条件下离心20min分离沉淀,调pH至中性,冷冻干燥后即得大豆11S球蛋白酸性亚基。pH8.0沉淀主要为碱性亚基,pH5.0沉淀主要为酸性亚基。
黄曲霉毒素B1抗原(AFB1-O-AS)的鉴定:
(1)抗原的紫外扫描
用甲醇配制1mg/mL的蛋白载体和抗原,黄曲霉毒素B1在200~400nm进行紫外扫描,根据吸收峰的变化来确定是否偶联成功。
如图4可知,黄曲霉毒素B1抗原紫外图谱中既表现出AS的紫外吸收特征,也呈现了AFB1的紫外吸收特征,表明AFB1和酸性亚基(AS)偶联成功。
(2)荧光光谱扫描。
将AFB1-O-AS和AFB1的浓度分别稀释到1.5mg/ml,在320nm处进行激发,然后采用荧光分光光度计算对酸性亚基(AS)和AFB1-O-AS进行荧光光谱分析。
如图5可知,AS在420nm处有荧光峰,可以判定为色氨基的荧光峰,由于AFB1-O的影响,AFB1-O-AS在420nm处的荧光强度明显下降,表明AFB1-O对酸性亚基(AS)具有一定的荧光猝灭作用,同时,AFB1-O-AS产生了450nm的荧光峰,可能为AFB1-O引入产生。
(3)ELISA测试
采用购买的标准黄曲霉毒素B1单克隆抗体对黄曲霉毒素B1抗原(AFB1-O-AS)进行ELISA测试。主要步骤如下:将黄曲霉毒素B1抗原(AFB1-O-AS),经过pH 9.6碳酸盐,4℃过夜包被,包被梯度分别为200倍、400倍、........、204800倍,包被体积为100微升,黄曲霉毒素B1单克隆抗体(仅与黄曲霉毒素B1抗原结构簇反应)含量为2mg/ml,稀释倍数为4万倍稀释,抗体用量为100微升,羊抗鼠HRP二抗稀释倍数为10000倍,一抗反应时间为40min,二抗反应时间为30min。
表1 直接法ELISA测试结果
由表1可知,实验结果表明黄曲霉毒素B1标准品经过半抗原改造后,经过间接ELISA测试,有明显的OD出现,说明黄曲霉毒素B1与大豆11S球蛋白酸性亚基载体进行了连接,抗原偶联成功。在0.625ppm包被条件下ELISA显色OD为1.701。
(4)间接竞争的ELISA测试
选择黄曲霉毒素B1单克隆抗体2.5万倍稀释,抗原稀释浓度为6400倍即0.625ppm,进行间接竞争elisa测试,标准品浓度为0、0.15ppb、0.3ppb、0.6ppb、1.2ppb、2.4ppb、4.8ppb。实验条件:与上面间接ELSIA测试条件基本一致(一抗反应时间,二抗浓度,二抗反应时间,底物反应时间),抗体与标准品溶液体积为50微升:50微升,实验结果如下:
表2 间接竞争ELISA测试结果
由表2可知,实验结果表明,黄曲霉毒素B1单克隆抗体与黄曲霉B1大豆蛋白抗原、黄曲霉B1标准品有竞争关系,说明黄曲霉毒素B1与大豆11S球蛋白酸性亚基载体进行了连接,抗原偶联成功,而且黄曲霉毒素B1单克隆抗体与大豆蛋白载体无交叉。
Claims (5)
1.一种黄曲霉毒素半抗原AFB1-O-CMO,其特征在于,分子结构式为:
2.根据权利要求1所述的一种黄曲霉毒素半抗原AFB1-O-CMO的制备方法,其特征在于,包括以下步骤:
(1)甲醇:双蒸水:吡啶溶液按体积比为4:1:1的比例混合后,再将黄曲霉毒素与半羧基羟胺盐酸CMO按质量比1:2混合后溶解于上述混合溶液中;
(2)对上述混合物80~90℃加热回流3~4h,直到所有的黄曲霉毒素完全转化为黄曲霉毒素半抗原;
(3)用旋转蒸发仪将溶剂旋干,加水后,用2mol/L的H2SO4调节PH=4,然后用5ml乙酸乙酯萃取2~3次,合并有机相,并用水洗涤至少2次,除去CMO,浓缩旋干液体,得到黄色油状半固态物质,即为黄曲霉毒素半抗原AFB1-O-CMO。
3.一种黄曲霉毒素人工抗原,其特征在于,所述人工抗原的分子结构式为:
所述载体蛋白Protein是大豆11S球蛋白酸性亚基。
4.根据权利要求3所述的一种黄曲霉毒素人工抗原的制备方法,其特征在于,采用EDC活性酯法合成,具体步骤如下:
(1)将黄曲霉毒素半抗原溶于2ml体积浓度为25%乙醇溶液中,称取4.5mg大豆11S球蛋白酸性亚基,溶于2ml纯水中,黄曲霉毒素半抗原与大豆11S球蛋白酸性亚基的投料摩尔比为60:1;
(2)加入50mg的EDC,室温下,避光搅拌反应48h;
(3)反应期间,再加入EDC各2次,每次50mg;
(4)反应产物在4℃搅拌下用PBS透析3天,每天换液3~6次,透析后得到纯化的黄曲霉毒素人工抗原AFB1-O-AS储存于-20℃。
5.根据权利要求4所述的一种黄曲霉毒素人工抗原的制备方法,其特征在于,所述大豆11S球蛋白酸性亚基的制备方法包括以下步骤:
(1)以1:5的体积比将大豆11S球蛋白溶于Tris-HCl缓冲液,加β巯基乙醇调节混合溶液的整体浓度至0.015mol.L-1,调pH为8.0,在90℃条件下水浴30min;
(2)4℃,10000r·min-1条件下离心20min,得到沉淀,调pH至5.0,冷冻干燥后即得大豆球蛋白碱性亚基;
(3)收集除去碱性亚基的上清液,调pH至5.0,4℃、6500r·min-1条件下离心20min分离沉淀,调pH至中性,冷冻干燥后即得大豆11S球蛋白酸性亚基。
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