文献类型：期刊文献

中文题名：酶联免疫吸附法快速检测畜禽产品中西马特罗药物残留

英文题名：Rapid detection of cimaderol residues in livestock and poultry products by enzyme linked immunosorbent assay

作者：司艳芳[1,2];岳锋[1];郭东光[1];齐永华[1];李鹏[1];孙国鹏[1];李新丽[2];马钰鑫[2];王选年[1]

第一作者：司艳芳

机构：[1]新乡学院,生物技术研究中心,新乡453000;[2]新乡学院3D打印学院,新乡453000

第一机构：新乡学院生命科学技术学院

年份：2022

卷号：13

期号：21

起止页码：6923-6931

中文期刊名：食品安全质量检测学报

外文期刊名：Journal of Food Safety and Quality

收录：CSTPCD;;北大核心:【北大核心2020】；

基金：国家自然科学基金项目(32102632);2018年国家重点研发计划项目(2018YFC1602902);河南省科技攻关项目(192102110066);河南省高等学校重点科研项目(19A230009);新乡市科技攻关项目(GG2019019)。

语种：中文

中文关键词：畜禽产品;西马特罗;重氮化法;兔源噬菌体抗体库;单链抗体;噬菌体展示

外文关键词：livestock and poultry products;cimatrol;diazotization method;rabbit phage antibody library;single-chain variable fragment;phage display

摘要：目的建立一种以单链抗体为基础的酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)快速检测畜禽产品中西马特罗(cimaterol,CIM)药物残留。方法以重氮化法合成的西马特罗完全抗原CIM-牛血清白蛋白(bovine albumin,BSA)免疫新西兰大白兔,用兔脾细胞总RNA反转录合成c DNA第一链,设计引物扩增兔源抗体重链可变区(heavy chain variable region,VH)和轻链可变区(light chain variable region,VL),通过45 bp的连接肽(Linker)使用新型重叠延伸聚合酶链式反应技术(splicing by overlap extension polymerase chain reaction,SOE-PCR)连接成VH-Linker-VL并大量制备;利用噬菌体表面展示技术将单链抗体(single-chain variable fragment,Sc Fv)片段与噬菌体展示载体p CANTAB-5e连接,电转化至大肠杆菌TG1感受态细胞,得到兔源噬菌体Sc Fv抗体库。通过聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)判断是否偶联成功,用紫外(ultraviolet,UV)扫描及基质辅助激光解析串联飞行时间质谱(matrix-assisted laser desorption tandem time-of-flight mass spectrometry,MALDI-TOF-MS)测定人工抗原的偶联比。用ELISA测定免疫兔血清质量。鉴定合成抗体库并对抗体库进行4轮淘选,取最优的CIM-Sc Fv进行氨基酸序列分析。结果CIM-BSA及CIM-鸡卵清白蛋白(ovalbumin,OVA)偶联比分别为8:1及10:1,免疫血清校价为1:32000,半数抑制浓度为9.77 ng/m L,标准曲线的拟合度为0.997。抗西马特罗兔源噬菌体Sc Fv免疫库的插入率为92%,库容为1×1010 pfu,多样性为83.3%,经淘选获得一株与CIM有结合活性的噬菌体Sc Fv菌。结论西马特罗完全抗原的合成效果良好,可用于建立西马特罗检测方法,兔源噬菌体抗体库为快速获得CIM单链抗体奠定基础,为西马特罗快速检测提供了新思路。
Objective To establish a single chain antibody based enzyme linked immunosorbent assay(ELISA)for rapid detection of cimaterol(CIM)residues in livestock and poultry products.Methods Immunized the New Zealand white rabbits with CIM-bovine albumin(BSA).The first strand of cDNA was synthesized by reverse transcription of total RNA from rabbit spleen cells.Merge primers were designed to amplify the heavy chain variable region(VH)and light chain variable region(VL)of rabbit antibody,which were connected into VH-Linker-VL through a 45 bp linker peptide by splicing by overlap extension polymerase chain reaction(SOE-PCR).Then prepared them in large quantities.Single chain variable fragment(ScFv)fragment was connected with phage display vector pCANTAB-5e by phage surface display technology,and electrically transformed into E.coli TG1 sensitive cells to obtain a rabbit-derived phage ScFv antibody library.The sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)was used to determine whether or not the success of coupling.Thus,the coupling ratio of artificial antigens was determined by ultraviolet(UV)scanning and matrix-assisted laser desorption tandem time-of-flight mass spectrometry(MALDI-TOF-MS).Serum quality in immune rabbits was determined by ELISA.The synthetized antibody library was identified and panned for four rounds.The optimal CIM-ScFv was selected for amino acid sequence analysis.Results The coupling ratios of CIM-BSA and CIM-ovalbumin(OVA)were 8:1 and 10:1,respectively.The CIM-BSA immunogen was immunized with New Zealand white rabbits.The calibration value of immune serum was 1:32000,and the median inhibition concentration was 9.77 ng/mL,and the fitting degree of the curve was 0.997.The insertion rate of the anti-cimaterol rabbit-derived phage ScFv immune was 92%,the capacity was 1×1010 pfu,and the diversity was 83.3%.A phage ScFv strain with binding activity to CIM was obtained by screened.Conclusion The synthesis effect of the complete antigen of CIM is favorable,and it can be used to establish the detection method of cimaterol.The rabbit-derived phage antibody capacity lays the foundation for the rapid acquisition of CIM single-chain variable fragment,and provides new ideas for the rapid detection of CIM.