氧氟沙星单链抗体的制备及间接竞争ELISA的建立  

Preparation of anti-ofloxacin single chain antibody fragment and establishment of indirect competitive ELISA method

作　　者：张运尚 王方雨[2] 胡曼 樊剑鸣[1] ZHANG Yunshang;WANG Fangyu;HU Man;FAN Jianming(College of Publie Healh,Zhenghou University,Zhengzhou 450001,China;Henan Provincial Key Laboratory of Animal Immunology,Henan Academy of Agriculture Sciences,Zhengzhou 450002,China)

机构地区：[1]郑州大学公共卫生学院,郑州450001 [2]河南农业科学院河南省动物免疫学重点实验室,郑州450002

出　　处：《黑龙江畜牧兽医》2021年第4期116-123,159,共9页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目“高适应性重组抗体库的构建、筛选及进化技术研究”(2018YFC1602902)。

摘　　要：为了制备高灵敏度、高特异性的抗氧氟沙星单链抗体并建立间接竞争酶联免疫吸附法(ELISA),试验采用噬菌体展示技术构建免疫小鼠噬菌体库,经过筛选后将分离出的阳性抗体转入大肠杆菌BL21中进行表达,然后采用分子对接的方法进行分子识别机制研究和虚拟突变,将关键氨基酸残基进行突变制备突变体,并建立间接竞争ELISA。结果表明:构建的噬菌体库经过四轮筛选后,最终库容量为2.35×10^(7) cfu/mL。从库中分离出25株单克隆阳性菌落,选择结合能力高的第22株单克隆菌落进行表达。分子对接后甲硫氨酸209(Met209)残基为单链抗体与氧氟沙星结合的关键氨基酸残基,将其突变为谷氨酰胺(Gln)后,总结合能从-5.32 kJ/mol降至-6.94 kJ/mol,表明突变体结合能力更强。建立检测方法的标准曲线方程为y=-0.192 7x+0.542 3 (R2=0.999 4),半数抑制浓度为1.66 ng/mL,相比于亲本单链抗体的21.08 ng/mL,敏感性提高了12.7倍。说明试验成功制备了高灵敏度、高特异性的抗氧氟沙星单链抗体并建立了间接竞争ELISA,为动物源性食品中氟喹诺酮类药物残留检测提供基础。In order to prepare high-sensitivity and high-specificity anti-ofloxacin single chain antibody fragment and establish an indirect competitive enzyme-linked immunoassay(ELISA),phage display technology was used in this experiment to construct a phage library of immunized mice. After screening, the isolated positive antibodies were transferred into E. coli BL21 for expression, and then molecular recognition mechanism research and virtual mutation were carried out by molecular docking method. The key amino acid residues were mutated to prepare mutants, and an indirect competitive ELISA detection method was established. The results showed that after four rounds of screening, the final library capacity of the constructed phage library was 2.35×10^(7) cfu/mL. Twenty five positive monoclonal colonies were isolated from the library, and the 22 nd monoclonal colony with high binding ability was selected for expression. The results of molecular docking showed that the amino acid residue Met209 was the key amino acid residue for the binding of single-chain antibody to ofloxacin. After it was mutated to Gln, the total binding energy decreased from-5.32 kJ/mol to-6.94 kJ/mol, indicating the mutant had stronger binding capacity. The standard curve equation to establish the detection method was y=-0.192 7x+0.542 3(R2=0.999 4),and the half inhibitory concentration was 1.66 ng/mL,which was 12.7 times in sensitivity higher than the parental single chain antibody fragment of 21.08 ng/mL. The results suggested that a high-sensitivity, high-specificity anti-ofloxacin single chain antibody fragment was successfully prepared in this experiment, and indirect compective ELISA method was established, which provides a basis for the detection of fluoroquinolone drug residues in animal-derived foods.

关 键 词：噬菌体展示技术 单链抗体 氟喹诺酮类药物 药物残留检测 分子识别机制 间接竞争酶联免疫吸附法 

分 类 号：S852.43[农业科学—基础兽医学] S859.796[农业科学—兽医学]