【摘要】：近年來,真菌毒素對食品的污染越來越嚴(yán)重,其中因玉米赤霉醇(ZER)所造成的污染已不容忽視。玉米赤霉醇及其代謝產(chǎn)物具有一定的雌激素作用,它隨食物鏈進(jìn)入機(jī)體后,會造成機(jī)體生長發(fā)育障礙,對機(jī)體第二性征產(chǎn)生影響。我國農(nóng)業(yè)部早在2002年就明確禁止玉米赤霉醇作為增重劑用于畜禽養(yǎng)殖。目前,用于食品及飼料中ZER的檢測方法主要有薄層色譜法、高效液相色譜法和免疫分析技術(shù)。薄層色譜法只能用于定性分析;高效液相色譜法的前處理過程復(fù)雜,需要專業(yè)人員操作,應(yīng)用大量的有毒有機(jī)溶劑,使其應(yīng)用受到一定的限制。而ELISA免疫分析技術(shù)具有快速、靈敏、簡便、特異等優(yōu)點(diǎn),正好可以彌補(bǔ)上述方法的不足,已被成功運(yùn)用于食品和飼料中真菌毒素的快速檢測。1、玉米赤霉醇人工抗原的合成及多抗血清的制備針對ZER第十六位上的羥基進(jìn)行改造,設(shè)計(jì)并合成半抗原ZER-16-羧丙基丁醚。分別采用混合酸酐法和碳二亞胺(EDC)法將改造后的半抗原與載體蛋白BSA和OVA偶聯(lián),制備出人工免疫抗原ZER-BSA和人工檢測抗原ZER-OVA。經(jīng)SDS-PAGE電泳鑒定,偶聯(lián)蛋白在凝膠上的遷移距離比載體蛋白小,故偶聯(lián)成功。之后免疫小鼠并成功制備出效價(jià)達(dá)1:2.56×104、IC50為15.77ng/m L的鼠源多抗血清。2、玉米赤霉醇單克隆抗體的制備及其特性鑒定成功獲得高效ZER多抗血清后,采用單克隆抗體技術(shù)和細(xì)胞融合技術(shù)將有效小鼠的脾細(xì)胞與SP/20骨髓瘤細(xì)胞進(jìn)行融合,多次亞克隆后篩選出了三株能夠穩(wěn)定分泌ZERm Ab的雜交瘤細(xì)胞株,分別為6B2E6、6B2E11和12B10A7,其細(xì)胞上清效價(jià)可達(dá)1:2.56×104、1:5.12×104和1:2.56×104。將敏感性最好的12B10A7細(xì)胞株注射到處理過的小鼠腹腔內(nèi),收集到的腹水效價(jià)為1:6.4×105。所得單抗經(jīng)鑒定為Ig G1型,親和常數(shù)Ka為4.7×1011L/mol,回歸方程為y=-0.3796x+0.7746,相關(guān)系數(shù)R2=0.9885,IC50=0.529ng/m L。ZERm Ab與黃曲霉毒素B1(AFB1)、伏馬菌素B1(FB1)等非同類毒素的交叉反應(yīng)率低,均小于0.5%。該細(xì)胞株不論是體外傳代還是凍存復(fù)蘇后都能穩(wěn)定地分泌抗體。3、ZER間接競爭ELISA檢測分析法的建立及在動物性食品中ZER殘留檢測的應(yīng)用通過反復(fù)的ELISA檢測試驗(yàn),確定了抗原最佳包被濃度為1:2000倍稀釋,抗體最佳工作濃度在1:10000倍稀釋,建立了ZER間接競爭ELISA檢測方法,用此方法對動物性食品中ZER殘留進(jìn)行檢測,回歸方程為y=-0.3582x+0.5949,相關(guān)系數(shù)R2=0.9925,IC50=0.184ng/m L�；厥章试�85%以上,變異系數(shù)小于10%,試驗(yàn)結(jié)果表明本試驗(yàn)建立的ZER間接競爭ELISA檢測分析法靈敏性強(qiáng),準(zhǔn)確率高,能夠有效地檢測食品中ZER的殘留。
[Abstract]:In recent years, the contamination of food caused by mycotoxins is becoming more and more serious, and the pollution caused by gibberellin (ZER) can not be ignored. Corn gibberellin and its metabolites have some estrogenic effects. When the food chain enters the body, it will cause the growth and development of the body and affect the secondary sexual characteristics of the body. As early as 2002, China's Ministry of Agriculture specifically banned gibberellin as a weight gain agent for livestock and poultry breeding. At present, the detection methods of ZER in food and feed mainly include thin layer chromatography, high performance liquid chromatography and immunoassay. TLC can only be used for qualitative analysis. The pretreatment process of HPLC is complex and requires professional operation. A large number of toxic organic solvents are used, so its application is limited to a certain extent. ELISA immunoassay has the advantages of rapidity, sensitivity, simplicity and specificity, which can make up for the shortcomings of the above methods. It has been successfully used in the rapid detection of mycotoxins in food and feed. The synthesis of artificial antigen of Zea gibberellin and the preparation of polyantiserum were modified to modify the hydroxyl group at the sixteenth position of ZER and to design and synthesize hapten ZER-16- carboxypropyl butyl ether. The modified hapten was coupled with the carrier proteins BSA and OVA by mixed anhydride method and carbodiimide (EDC) method, respectively. The artificial immune antigen (ZER-BSA) and the artificial detection antigen (ZER-OVA.) were prepared. SDS-PAGE electrophoresis showed that the transfer distance of the conjugated protein on the gel was smaller than that of the carrier protein, so the coupling was successful. Then mice were immunized and the mouse polyclonal antisera with the titer of 1: 2.56 脳 104 IC50 as 15.77ng/m L were successfully prepared. After the preparation and characterization of monoclonal antibody to Zea gibberellin, the highly effective ZER polyclonal antibody was obtained. The effective mouse spleen cells were fused with SP/20 myeloma cells by monoclonal antibody technique and cell fusion technique. After several subclones, three hybridoma cell lines were selected to secrete ZERm Ab stably. The supernatant titers of 6B2E6B2E11 and 12B10A7 were 1: 2.56 脳 10 ~ 4 ~ 1: 5.12 脳 10 ~ 4 and 1: 2.56 脳 10 ~ 4 脳 10 ~ 4 respectively. The most sensitive 12B10A7 cell line was injected into the peritoneal cavity of the treated mice. The titer of ascites collected was 1: 6.4 脳 105. The McAbs were identified as Ig G1 type, the affinity constant Ka was 4.7 脳 1011L / mol, the regression equation was y1-0.3796x 0.7746, and the correlation coefficient was R2O0.9885C500.29ngr / m L.ZERm Ab with aflatoxin B1 (AFB1), fumonisin B1 (FB1) and other non-congener toxins, all of which were less than 0.5mg / m. This cell line can secrete antibody. 3ZER indirectly competitive ELISA assay and its application in animal food by repeated ELISA detection test, no matter whether it is passaged in vitro or after cryopreservation and resuscitation. The best coating concentration of antigen was 1: 2000 times dilution and the best working concentration of antibody was 1: 10 000. An indirect competitive ELISA method for the detection of ZER residues in animal food was established by using this method. The regression equation was yr 0.3582x 0.5949, and the correlation coefficient was R2O 0.9925IC500.184ng/ mL. The recovery was more than 85%, and the coefficient of variation was less than 10. The results showed that the indirect competitive ELISA method established in this study was sensitive and accurate, and could be used to detect ZER residues in food effectively.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：TS207.5;S816.17

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 薄存香;張振玲;郭啟明;劉衍忠;李莉;謝琳;;玉米赤霉醇對大鼠的胚胎毒性[J];癌變.畸變.突變;2006年03期

2 何慶華;劉仁榮;許楊;;利用噬菌體肽庫淘選玉米赤霉烯酮的模擬表位[J];食品科學(xué);2007年08期

3 王昕;姚佳;果旗;里南;江帆;王雄;張建新;;IAC-HPLC法檢測牛奶中黃曲霉毒素和玉米赤霉醇及類似物質(zhì)量分?jǐn)?shù)[J];中國乳品工業(yè);2014年06期

，

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