【作者】 梁晓；

【导师】 张素霞； 王战辉；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2014， 博士

【摘要】 磺胺类药物(sulfonamides, SAs)是一类广泛应用于畜牧业的广谱抗菌药物,但由于其不合理使用甚至滥用造成了动物源食品中药物残留问题,影响食品安全,进而危害人类健康。建立快速、高效、广谱、可靠的检测方法监控动物源食品中SAs残留,对食品安全和人类健康保障具有重要意义。本文以磺胺类药物受体蛋白二氢叶酸合成酶(Dihydropteroate Synthase, DHPS)为研究对象,建立快速检测方法检测牛奶中磺胺药物多残留。体外扩增来源于肺炎链球菌R6和大肠杆菌ATCC25922的flop基因,分别构建表达载体flop-pET28b,优化诱导表达条件,发现诱导温度20℃、诱导时间14～17h和0.5mM异丙基-β-D-硫代吡喃半乳糖苷(Isopropyl β-D-1-Thiogalactopyranoside, IPTG)时诱导效果最佳。采用镍柱亲和层析和离子交换方法纯化DHPS蛋白,通过纯化条件优化,发现100mM咪唑为最适洗脱缓冲液浓度。100mL大肠杆菌培养物可获得约3mg目的蛋白。采用二环己基碳二亚胺(Dicyclohexylcarbodiimide,DCC)法偶联辣根过氧化物酶(Horseradish Peroxidase,HRP)与磺胺类药物及其衍生物,获得7种酶标记物,其中4-(4-氨基-苯磺酰胺基)苯甲酸(4-(4-amino-benzenesulfonylamino) benzoic acid,CS)-HRP与DHPS结合力最强,对磺胺二甲基嘧啶(Sulphamethazine, SM2)的灵敏度最高。以CS-HRP与来源于肺炎链球菌的DHPS组合建立微孔板法检测SAs多残留,进行了条件优化,分别以31种磺胺类药物及其类似物建立标准曲线,其中29种磺胺类药物的IC50(50%结合时待测物浓度)值为2.95～56.22ngmL-1,均低于最高残留限量(Maximum Residue Limit,MRL)。以CS-HRP与来源于大肠杆菌的DHPS组合建立微孔法检测SAs多残留,29种磺胺类药物的IC50值为6.16～182.27ng mL-1,选择CS-HRP与来源于肺炎链球菌的DHPS组合建立微孔板法检测牛奶中SAs多残留,纯牛奶稀释5倍、脱脂牛奶稀释3倍即可用于检测。纯牛奶中磺胺间甲氧嘧啶(Sulfamonomethoxine,SMM)、磺胺喹噁啉(Sulfaquinoxaline,SQX)、SM2、磺胺甲基异嗯唑(Sulfamethoxazole,SMZ)和磺胺二甲氧嘧啶(Sulfadimethoxine,SDM)的添加回收率为83.3～108.8%,变异系数为1.1～15.8%；脱脂奶中的添加回收率为72.7～129.3%,变异系数为3.0～16.0%。为简化快速检测方法,用焦磷酸盐(pyrophosphate,PPi)取代DHPS的第一底物6-羟甲基-7,8二氢蝶呤焦磷酸盐(6-hydroxymethyl-7,8-dihydropterin pyrophosphate, DHPPP),以CS-HRP与来源于肺炎链球菌的DHPS组合建立SAs残留微孔板检测方法,进行了条件优化,其中9种SAs和PABA的检测限(limit of detection,LOD)低于MRL值,28种磺胺类药物的IC50值为426～50000ngmL-。合成了7种荧光标记物,以来源于肺炎链球菌的DHPS和最佳荧光标记物CS-BDF组合建立检测牛奶中SAs多残留的荧光偏振分析方法(Fluorescence Polarization Immunoassay, FPIA).进行了条件优化,29种SAs的IC50值为4.90～108.24ng mL-1, PABA的IC50值最低,为3.80ng mL-1。牛奶中SMM、SQX、SM2、SMZ和SDM的回收率为53.3～101.9%,变异系数为0.29-19.7%。为了深入了解DHPS与磺胺类药物的相互作用,为方法灵敏度的提高和DHPS体外进化提供理论基础,本研究利用Surflex-X-2.0对DHPS与6种SAs进行分子对接研究,并给出评分,相对亲和力的变化趋势与评分结果基本一致。DHPS与SAs作用的2个关键氨基酸残基为Ⅱe272和Asp221,疏水作用力是其相互作用的决定因素。综上所述,本研究获得了磺胺类药物受体蛋白DHPS,其对磺胺药物的亲和力高、广谱性好,可代替抗体应用于快速检测方法建立。更多还原

【Abstract】 Sulfonamides (SAs) are a group of synthetic antibiotic agents that are widely used in veterinary practice for the treatment of infectious diseases and as growth-promoting feed additives. Illegal use of SAs or non-compliance of animal-treatment protocols may cause the residues of antibiotics in animal-derived food. It seriously infects food safety and human health, so it is necessary to monitor these chemical residues in food samples in order to obtain a better food safety assurance. For SAs residues, the screening methods must be precise, high sensitive and cheap, moreover, these methods must be simple, quick and broad-spectrum as it possible. In this paper, Dihydropteroate Synthase (DHPS) was produced and rapid detection assay based-on DHPS was developed for SAs in milk.The gene flop encoding DHPS from Streptococcus pneumonia R6and Escherichia coli ATCC25922was ampilied using PCR and inserted in expression vector pET28b, respectively. After optimization, expression was carried out at20℃for14～17h after0.5mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG) was added. Purification of DHPS required two steps of Ni2+chelation affinity chromatography (100mM imidazole in elution buffer) and ion exchange.To identify the ideal Horseradish Peroxidase (HRP)-conjugate matched with DHPS, seven haptens were conjugated to HRP by DCC (dicyclohexylcarbodiimide) method, respectively. Among these conjugates, CS-HRP showed the highest affinity to DHPS and the highest sensitivity to detect sulphamethazine (SM2), and was thus used to develop and optimize the direct competitive assay. We develop a microplate assay based on CS-HRP and DHPS from Streptococcus pneumonia R6for SAs, and after optimization29SAs used in the study could be detected with IC50values ranging from2.95ng mL-1to56.22ng mL-1which were under the Maximum Residue Limit (MRL). IC50values for29SAs ranged from6.16ng mL-1to182.27ng mL-1in the microplate assay based on CS-HRP and DHPS from Escherichia coli ATCC25922. DHPS from Streptococcus pneumonia R6was slected due to high sensitivity obtained. Five SAs, sulfamethoxazole (SMZ), sulfadimethoxine (SDM), sulfaquinoxaline (SQX), sulfamonomethoxine (SMM), and SM2were spiked in whole milk and skim milk samples with simple pretreatment. The recoveries were83.3%～108.8%with coefficient of variation (CV) of1.1%～15.8%and72.7%～129.3%with CV of3.0%～16%in whole milk and skim milk, respectively. In order to simplify the assay, PPi (pyrophosphate) was used to replace the first substrate of DHPS,6-hydroxymethyl-7,8-dihydropterin-pyrophosphate (DHPPP). Under optimized conditions,9SAs and PABA could be detected below100ng mL-1and28SAs used in the study could be detected with IC50values ranging from426ng mL-1to50000ng mL-The best fluorescent tracer CS-BDF was produced and a fluorescence polarization immunoassay (FPIA) based on DHPS from Streptococcus pneumonia R6for SAs in milk was developed. The IC50values of29SAs ranged from4.90ng mL-1to108.24ng mL-1and the lowest IC50value (3.80 ng mL-1) was for PABA. Analysis of SMZ, SDM, SQX, SMM, and SMX fortified milk samples by the FPIA showed average recoveries ranging from53.3%to101.9%with CV0.29～19.7%.In order to increase the sensitivity of the assay of fast detection further more and provide the theoretic foundation for vitro protein evolution, we investigated the binding between6SAs and DHPS using Surflex-X-2.0. The correlation between relative binding affinitys (RBAs) and docking score was investigated, and good correlation was observed. Ile272and Asp221were the important amino acid sites in the interaction between DHPS and SAs, and hydrophobic effect was a decisive factor in the interaction between DHPS and SAs. In conclusion, we produed DHPS with high affinity and broad-spectrum activity for SAs and ordinary antibody could be replaced by DHPS for SAs detection.更多还原