【作者】 黄运红；

【导师】 曹郁生； 许杨；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2013， 博士

【摘要】 食品和生态环境中的磺胺类药物残留,不仅危害人类键康,而且造成巨大经济损失,越来越受到人们的关注。建立磺胺嘧啶的快速、准确检测方法对食品安全,人类健康以及打破发达国家技术壁垒具有重要意义。论文针对这一急需解决的重要问题,制备了磺胺嘧啶人工抗体(Artificial antibody),建立了分子印迹柱-高效液相色谱法检测食品样品中磺胺嘧啶残留的方法。同时进行了磺胺嘧啶单链抗体(Single-chain variable fragment, scFv)的研究,构建了单链抗体随机突变文库,以期从分子水平探讨更新、更快速准确的方法。主要研究结果如下：1.采用紫外吸收光谱法和HNMR实验的方法研究了磺胺嘧啶模板分子和丙烯酰胺功能单体之间的相互作用。结果表明,磺胺嘧啶和丙烯酰胺分子间会形成氢键作用,并随着磺胺嘧啶浓度的增加,丙烯酰胺混合液的紫外吸收会发生红移现象且吸收峰的强度减弱。2.采用本体聚合法制备了磺胺嘧啶分子印迹聚合物,并采扫描电子显微镜法、傅立叶变换红外光谱分析法、Brunauer-Emmett-Teller实验方法、及吸附动力学实验等方法研究磺胺嘧啶分子印迹聚合物的特性。结果表明,MIP表面含有大量模板分子被洗脱后所留下的立体空穴,MIP的比表面积为182.2m2g-1,MIP的吸附容量为7.8mg/g。3.采用单因素实验方法优化磺胺嘧啶分子印迹柱的固相萃取条件。结果表明,在2.5%乙腈-水溶液(v/v)为萃取液,流速为2mL/min,40%乙腈-水溶液(v/v)为洗脱液,洗脱体积为3mL最佳的条件下,磺胺嘧啶分子印迹柱的萃取效率为99%。4.建立了分子印迹柱-高效液相色谱检测猪肉中磺胺嘧啶残量的方法。结果表明,在最佳固相萃取条件下,猪肉样品中磺胺嘧啶的浓度在50-4000μg／kg范围内,检测线性关系良好,线性回归方程为y=0.063x+0.0294,(R2=0.9998,n=3),定量限为33.3μg/kg,检出限为10μg/kg,加标回收率≥75.6%,相对标准偏差≤6.1%。5.采用RT-PCR技术与SOE-PCR技术构建了磺胺嘧啶单链抗体的基因,并采用生物信息学方法分析磺胺嘧啶单链抗体的结构特性。结果表明,磺胺嘧啶单链抗体的重链和轻链的可变区均含有鼠源的3个CDR区和4个FR区；NCBI DNA Blast未见相同序列,但重链可变区基因与鼠源的IGHV1-14*01的同源率达83.7%,轻链可变区基因与鼠源的IGKV6-20*01同源率达91.6%；单链抗体氨基酸序列提交至ExPASy分析可知,单链抗体的分子量为26582.4,分子式为C1174H1779N3130376S9,等电点为7.6,亲水性指数为-0.484,不稳定系数为49.38,该单链抗体属于不稳定蛋白。6.采用分子生物技术与基因克隆技术构建了磺胺嘧啶单链抗体的原核表达载体pET22b-SD,并将其转化至E.coli BL21(DE3)中,采用单因素实验方法优化了重组菌株E.coli BL21(DE3)的表达条件。结果表明,重组菌株E.coli BL21(DE3)以包涵体形式表达了磺胺嘧啶单链抗体；诱导表达的最佳条件为诱导温度为30℃、IPTG诱导浓度为300μM、起始pH7、装料系数为30%和诱导时间为8h；蛋白印迹和酶联免疫吸附实验表明磺胺嘧啶单链抗体具有生物活性。7.采用基因克隆技术将磺胺嘧啶单链抗体基因克隆至分泌型pPIC9K表达载体中,并转化至毕赤酵母GS115。结果表明,MM平板和MD平板筛选获得Muts型毕赤酵母GS115重组子,含G418抗生素的YPD平板筛选获得多拷贝毕赤酵母GS115重组子；在甲醇诱导下,多拷贝毕赤酵母GS115重组子表达了磺胺嘧啶单链抗体。8.采用易错PCR技术构建了磺胺嘧啶单链抗体随机突变噬菌体文库。结果表明,在Mg2+浓度为7mmol/L, dATP和dGTP的浓度分别为0.2mmol/L, dCTP和dTTP的浓度分别为1mmol/L, Mn2+浓度为0.3mmol/L最佳易错PCR条件下,基因突变率为1.3%,突变具有一定程度的A／T偏向性；噬菌体文库库容为1.26×106。更多还原

【Abstract】 Much attention has been pay to the hazard of sulfadiazine (SD) residues in foods and ecological environment, for these residues can cause serious problems including human health and huge economic losses. It is important to develop a rapid and accurate method for detection of SD. In this dissertation, the artificial antibody against SD was prepared by the molecular imprinting technology. Sulfadiazine residues in the pork were detected by the molecular imprinted column coupling with high performance liquid chromatography method. Single-chain variable fragment against SD was prepared by the modern molecular biotechnology and the gene engineering technology. The phage library which the gene of single-chain variable fragment against SD mutated was constructed. The main research results are as follows:1. Such association between the functional monomer and the template molecule has been confirmed by the Ultra Violet absorption spectra and nuclear magnetic resonance spectroscopy (’HNMR). It was clearly observed that the decreased absorption peak of the mixtures and the redshift effect were attributed to an association between the monomer and template molecules via hydrogen bonding. In the mixture solution, the amino group (-NH2) and the carbanyl group (C=O) in a molecule of Acrylic acid (AM) can interact with the functional group (amino group and carbanyl group) of the template molecule, leading to the formation of the precursor through hydrogen bonding.2. The Molecularly imprinted polymers (MIP) for SD was synthesized using bulk polymerization. The characters of MIP were described by the scanning electron microscopy (SEM), the Fourier transform infrared spectrometry, Brunauer-Emmett-Teller (BET), and the adsorption kinetic experiment. It was noted that the surface of the MIP exhibited more porous structure, the BET surface area of the MIP was182.2m2g-1, and the adsorption amount of the MIP for SD reached7.8mg/g. The phenomenons showed that the MIP could be successfully synthesized using bulk polymerization.3. The molecularly imprinted solid-phase extraction (MISPE) column was selected as an extraction device. To obtain the optimum extraction efficiency, several parameters related to the molecular imprinted column, including column solvent, extraction flow-rate, eluent of the sample matrix and eluent volume, were investigated. The recovery of SD was99%under the optimum conditions which the acetonitrile with2.5%of the volume fraction was selected for the column solvent, the flow-rate of column solvent was2mL/min, the mixture of acetonitrile and water (2/3, v/v) solution was the optimum eluent, and the3mL of eluent was selected for the eluent volume.4. To test the SD response linearity, a series of standard solutions of SD in the concentration range50-4000μg kg-1were analyzed, using blank sample extraction solvent. The relationship between peak area (y) and sample concentration (x) was linear for SD according to the equation y=0.063x+0.0294,(R2=0.9998),(n=3). The limit of detection (LOD) and the limit of quantification (LOQ) are10.0μg kg-1and33.3μg kg-1respectively. It can be seen that the recovery of SD in the spiked pork samples treated by the MISPE column are more than75.6%, and the relative standard deviation (RSD) values of them are found to be less than6.1%. The proposed method has been successfully applied to monitoring sulfadiazine residues in pork.5. The gene of scFv against SD was prepared by the reverse transcription-polymerase chain reaction (RT-PCR) technology and the splicing by overlap extension PCR (SOE-PCR) technology. The phy-chemical properties of scFv against SD were analyzed through the bioinformatics methods. The heavy chain and light chain of single-chain variable fragment against sulfadiazine hold four FR and three CDR in the antibody from Murine, respectively. It was not found that the sequence of single-chain variable fragment against sulfadiazine was not the same sequence in the NCBI DNA Blast. The homologous rate was83.7%between the gene of heavy chain and IGHV1-14*01. The homologous rate was91.6%between the gene of light chain and IGKV6-20*01. It is known that the molecular weight is26582.4, the molecular formula is C1174H1779N313O376S9, the isoelectric point is7.6, the hydrophilic index is-0.5484, and the instability index is49.38by ExPasy software. The scFv against SD belongs to instability protein. 6. The prokaryotic expression vector pET22b-SD with the gene of the scFv against SD was prepared by the molecular biotechnology and the gene engineering technology. The expressed conditions were optimized by the single factor experiment mothed. The scFv against SD were expressed as inclusion bodies in E.coli BL21(DE3). The results showed the optimum induced conditions that the induced temperature was30℃, the concentration of IPTG was300μM, the pH was7, the coefficient of charge was30%, and the expression time was8h. Western blotting and Enzyme-linked immunosorbent assay (ELISA) confirmed that the scFv against SD expressed in the E.coli BL21(DE3) had bioactivity.7. The gene of scFv against SD was cloned to the secreting type expression vector pPIC9K. The expression vector pPIC9K-SD was transformed to Pichia pas tor is G115. The Muts type of Pichia past or is GS115recombinants were screened by both MM and MD plates. Multi-copy Pichia pastor is GS115were screened by the YPD agar plates containing G418. The scFv against SD in the Pichia pas tor is GS115was successfully secreting expressed under the methanol induction.8. The gene of the scFv against SD was random mutated by the error-prone PCR technology. The mutation rate was1.3%under the optimal conditions that the concentration of Mg2+was7mmol/L, the concentration of dATP and dGTP was0.2mmol/L respectively, the concentrations of dCTP and dTTP was1mmol/L respectively, and the concentration of Mn2+was0.3mmol/L. A/T mutation rate was higher than G/C mutation rate. The storage capacity of phage library which the gene of the scFv against SD was mutated was1.26x106.更多还原