【作者】 熊啸；

【导师】 熊勇华；

【作者基本信息】 南昌大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 黄曲霉毒素（Aflatoxin，简称AF）是真菌的次级代谢产物，主要由黄曲霉、寄生曲霉和特曲霉产生，广泛存在于花生、棉籽、玉米、小麦和稻米等农产品中。因AF具有强烈的毒性和致癌性，1993年AF被世界卫生组织（WHO）的癌症研究机构划定为Ⅰ类致癌物。AF是一类结构类似化合物，已经分离鉴定出黄曲霉毒素B₁（Aflatoxin B1，简称AFB₁）、B₂、G₁、G₂、M₁、M₂等二十余种，其中以AFB₁毒性最强，AFM₁毒性虽然低于AFB₁，然而与氰化钾和砒霜相比，仍属剧毒物质，为强致癌剂。为了防止AFM₁含量超标的食品尤其是乳制品进入人类食物链，许多国家已经制定了食品中AFM₁的限量标准。因此，建立一种快速检测AFM₁的方法具有重要意义。本论文研究的内容主要包括以下两个方面：1．黄曲霉毒素M₁直接竞争ELISA的建立（1）制备AFM₁肟化物，然后通过活泼酯法将AFM₁肟与辣根过氧化物酶进行偶联。（2）通过棋盘滴定法确定抗AFM₁单抗与AFM₁-HRP的最佳工作浓度，然后以该浓度为基础建立AFM₁直接竞争ELISA标准曲线，最低检测下限为0.1ng／mL，线性范围是0.1 ng／ml至4 ng／ml，IC₅₀是2.2 ng／ml。2．采用噬菌体随机展示肽库淘选AFM₁模拟表位，（1）以抗AFM₁单克隆抗体为靶分子，从噬菌体随机7肽库中进行4轮亲和淘选。从第4轮噬菌平板上随机挑选21个噬菌斑，经间接ELISA和间接竞争ELISA鉴定，获得了4个能与抗AFM₁单克隆抗体结合并且不和封闭液发生交叉反应的阳性噬菌体克隆，这4号噬菌体均能被黄曲霉毒素M₁抑制，分别编号为A1，A2，A3，A4。（2）提取上述4个噬菌体的DNA，进行测序。根据噬菌体文库简要遗传密码将插入的DNA序列翻译成氨基酸序列。A1，A2两号噬菌体插入片段氨基酸序列为LTSFPRH，A3，A4两号噬菌体插入片段氨基酸序列为MAPSSWR。（3）用上述A1与A3噬菌体替代AFM₁-HRP抗原分别建立间接竞争ELISA标准曲线，结果显示两曲线相似，曲线线性范围为0.1-5ng／mL，IC₅₀为3ng／mL，最低检测限度为0.1ng／mL。更多还原

【Abstract】 Aflatoxins （AF） are toxic secondary metabolites mainly prouduced by the fungiAspergillus flavus, Aspergillus parasiticus and Aspergillus nomius on foods andfeedstuffs. AF has been evaluated by the International Agency for Ressarch on Cancerof WHO as a classⅠhuman carcinogen in 1993 because of their toxic andcarcinogenic potentials to humans and animals. Among 20 different types ofaflatoxins identified, the major ones are aflatoxin B₁ （AFB₁）, AFB₂, AFG₁, AFG₂,AFM₁, AFM₂ and so on, and AFB₁ is the most potent hepatocarcinogen known inmammals. Although AFM₁ is not as toxic as AFB₁, but compared with Potassiumcyanide, it still has strong toxic power. Many countries have established regulatorylimits for AFM₁ in foods. In order to avoid foods and foodstuffs which are highcontaminated by AFM₁ especially dairy products enter human food chain. So it isvery important to develop convenient operation method for rapid detecting AFM₁.The main results are as follows:1. The establishments of AFM₁ direct competition ELISA（1） AFM₁ oxime has to be synthesized first, then conjugate AFM₁ with HRP.（2） The best working concentration of AFM₁-HRP and anti AFM₁ monoclonalantibody were optimized, and the direct competitive ELISA standard curve wasestablished basis on the above conditions, the linear range of the inhibition curve wasfrom 0.1 ng/ml to 4 ng/ml. The IC₅₀ was 2.2 ng/ml and the detecting limit was 0.1ng/ml.2. Panning mimotopes of aflatoxin M₁ by phage display peptide library（1） The monoclonal antibody against the aflatoxin M₁ was used as ligand to panthe binding peptide from the Ph.D.-7^TM phage display peptide library. After fourrounds of panning, 20 clones were picked out randomly from the fourth round. Thosephage plaques were amplified and purified, respectively. Four of clones wereidentified positive by indirect ELISA. They were screened by indirect competitiveELISA and named phageA1, A2, A3, A4.（2） DNA of four phage clones was extracted, sequence and then translated into the amino acid sequence according to the genetic code. The results showed that theyrepresent 2 different sequencing （A1 and A2, A3 and A4 are the same sequencing）.The peptide sequences of 4 phage clones were analyzed by sequencing and displayed.The peptide sequence of A1 and A2 is LTSFPRH, the peptide sequence of A3 and A4is MAPSSWR（3） The indirect competitive ELISA curves were established and the linear rangeof the inhibition curves were between 0.1 ng/mL and 5 ng/mL, IC₅₀ was 3 ng/mL, thedetecting limit was 0.1 ng/mL.更多还原