【作者】 邢丽杰；

【导师】 罗小玲；

【作者基本信息】 石河子大学 ， 农产品加工与贮藏， 2008， 硕士

【摘要】 蓖麻毒素（Ricin Toxion, RT）是一种核糖体失活蛋白,由A、B链组成,小鼠LD50仅为7-10μg/kg。目前国内外对蓖麻毒素的研究多为用于抗肿瘤的免疫毒素的制备以及中毒机理的研究,由于蓖麻毒素的毒性高,从摄入到死亡仅需1²h,致死量极小,感官无法辨认,蓖麻毒素类毒素和抗毒素正在研究中,目前尚无用于人的特效药,因此,蓖麻毒素的快速检测,特别是在被人体摄入之前就能够高效灵敏的检测到,就显的极为重要。本试验将制备的抗蓖麻毒素腹水单抗纯化后进行抗原表位的分析,结果显示4D8、4D12、1H4三株单抗的抗原决定簇均位于蓖麻毒素A链上。对4D8标记了辣根过氧化物酶和FITC,基于表面等离子共振仪与流式细胞仪分析后,分别建立了夹心ELISA、IMS-ELISA、SPR与液态芯片检测蓖麻毒素的方法。1.夹心ELISA检测蓖麻毒素方法的最佳条件为:45ug/ml抗RT的多克隆抗体为捕获抗体、1:4000倍稀释的HRP-4D8为检测抗体、5%脱脂乳+2%PEG20000为封闭剂,洗去检测抗体后用TMB在37℃下显色20min,测定OD₄₅₀;该方法不跟与蓖麻毒素结构相近的其它几种毒素发生交叉反应,最低检出浓度为5ng/ml,线性范围为0.1-1ug/ml,对纯净水的回收率为107.6%。2. IMS-ELISA检测蓖麻毒素方法的最佳条件为:将多抗与磁珠偶连并封闭后用PBST缓冲液悬浮在EP管中,加入抗原后37℃轻微振荡孵育1h,每15min吹打重悬一次,清洗后加入4000倍稀释的HRP-4D8检测抗体200μl孵育,清洗后加TMB底物在37℃下显色20min,测定OD₄₅₀;该方法不跟与蓖麻毒素结构相近的其它几种毒素发生交叉反应,最低检出浓度为50ng/ml,当RT浓度介于0.2-10ug/ml之间时,呈现极好的线性关系,对纯净水、秋梨膏、纯牛奶的回收率分别为95%、93%、68%。3.由SPR动力学分析了四株抗蓖麻毒素单抗的亲和性:并建立了SPR检测蓖麻毒素与相思子毒素的SPR检测方法,对蓖麻毒素的线性范围为0-2000ng/ml之间,最低检出浓度为0.05ng/ml,对相思子毒素的线性范围为250ng/ml-2000ng/ml,最低检出浓度为0.5ng/ml。对两种毒素的混合物同时检测时与单独检测的效果一样。4.本研究同时基于流式细胞仪与微球载体建立了对蓖麻毒素的检测方法,对蓖麻毒素的线性范围为10-1000ng/ml之间,最低检出浓度小于0.1ng/ml。5.采用PCR法扩增出蓖麻毒素B链基因,并将其与pMD18-T克隆载体相连接,经序列分析正确后插入到表达载体pET-28a中,构建重组表达质粒pET-28a-RTB,将构建好的重组质粒转化入BL21感受态细胞中,经IPTG于37℃诱导表达后,得到的融合表达的目的蛋白,经SDS-PAGE分析,相对分子量约为31KD,利用Western bloot与间接ELISA鉴定证明其具有反应原性,为制备RTB单克隆抗体及提高检测方法的敏感性打下基础。更多还原

【Abstract】 This paper reported that 4D8、4D12 and 1H4 are McAb anti-ricin, which purificated by protein-G chromatographic column, and ELISA data showed that their antigenic determinant were located on ricin A china. we labered 4D8 with horse radish peroxidase（HRP） and FITC respectively, then established ELISA、IMS-ELISA、surface plasma resonance（SPR） to detect ricin. test data indicated:1.The optimum condition of sandwich ELISA assays for ricin is: diluted RT polyclonal antibody to 45 ug/ml as capture Ab, diluted HRP-4D8 to 4000 times as detection Ab, chose 5%skimmed milk+2%PEG20000 as blockage, add TMB substrate solution after wash away detection Ab, incubation at 37℃for 20min. read OD₄₅₀. This method don’t have cross-reaction with other resemble toxion, minimum detectable concentration is 5ng/ml, The detection range is 0.1-1ug/ml, standard recovery rate to purified water is 107.6%.2.The optimum condition of IMS-ELISA assays of ricin is: coupling beads with polyclonal antibody anti-ricin, add PBST in EP tube to diluted beads after block, add assay sample incubate for 1h at 37℃with slow tilt rotation. Resuspension with micropipettor every 15min, diluted HRP-4D8 to 4000 times, add 200μl as detection Ab, add TMB substrate solution 100μl after wash away detection Ab, incubat at 37℃for 20min. read OD₄₅₀. This method don’t have cross-reaction with other resemble toxion, minimum detectable concentration is 50ng/ml, The detection range is 0.2-10ug/ml, standard recovery rate to purified water、pear jam and milk respectively is 95%、93%、68%.3.Kinetic analysis base on SPR result showed the affinity to ricin:4D8 KA=3.16×10¹² ,KD=3.16×10^-12;4D12 KA=1.53×10¹³, KD=6.55×10^-14;2F KA=4.1×10⁸, KD=2.44×10^-9;1C KA=1.86×10¹⁰, KD=5.38×10^-11. Establish the metherd base on SPR assay for ricin and abrin, The detection range for ricin is 0-2000ng/ml, for abrin is 250-2000ng/ml; minimum detectable concentration for ricin is 0.05ng/ml, for abrin is 0.5ng/ml; And the effect of detect single toxion is equal to mixture of ricin and abrin.4. Establish the metherd base on FCM and beads assay for ricin. The detection range for ricin is 10-1000ng/ml, minimum detectable concentration for ricin is 0.1ng/ml.5. The DNA of RTB was cloned from the ricin DNA by PCR and inserted into the carrier pMD18-T, The fusion expression vector RTB-pET28 was contructed and transformed to E.coli BL21 after sequenced wand. The expressed fusion protein after induction with IPTG at 37℃. SDS-PAGE and Western blotting analysis showed that the 31KD fusion protein was expressed correctly in E.coli. and the bioactivity was detected by indirect ELISA. The results provided reliable basis for the specificity of ELISA detect ricin.更多还原