摘要
为了制备高灵敏度、高特异性的抗新霉素单链抗体,试验采用从杂交瘤细胞中提取单链抗体基因,转入大肠杆菌表达系统进行表达。然后采用分子对接的方法研究抗新霉素单链抗体和新霉素分子的识别机制并进行虚拟突变,将关键结合位点的氨基酸残基进行突变,在对突变体进行表达和纯化后建立间接竞争酶联免疫吸附分析方法。结果表明:氨基酸残基Leu227为单链抗体与新霉素结合的关键氨基酸残基,将其突变为Asp,获得突变体。IC 50为1.58 ng/mL,相比于亲本单链抗体的26.23 ng/mL,突变体的敏感性提高了16.6倍。试验成功制备了高灵敏度、高特异性的抗新霉素单链抗体,为动物源性食品中的抗生素残留检测提供了研究基础。
In order to prepare high-sensitivity and high-specificity anti-neomycin single-chain antibodies,the experiment used genes extracted from hybridoma cells and directly transferred to E.coli expression system for expression.Then,the molecular docking method was used to study the recognition mechanism of anti-neomycin single-chain antibody and neomycin molecule and virtual mutation was performed.The amino acid residues of the key binding site were mutated,and the indirect competitive enzyme-linked immunosorbent assay method was established after expression and purification of the mutant.The results showed that the amino acid residue Leu227 was the key amino acid residue for the binding of single-chain antibody to neomycin,and it was mutated to Asp to obtain a mutant.The IC 50 was 1.58 ng/mL,which was 16.6 times more sensitive than the parental single-chain antibody(26.23 ng/mL).It showed that the test successfully prepared high-sensitivity and high-specificity anti-neomycin single-chain antibody,which provided a research basis for the detection of antibiotic residues in animal-derived foods.
作者
王方雨
张运尚
胡曼
樊剑鸣
WANG Fang-yu;ZHANG Yun-shang;HU Man(Key Laboratory of Animal Immunology,Henan Academy of Agriculture Sciences,Zhengzhou,Henan 450002;College of Public Health,Zhengzhou University,Zhengzhou,Henan 450001)
出处
《安徽农业科学》
CAS
2020年第20期103-107,150,共6页
Journal of Anhui Agricultural Sciences
基金
国家重点研究计划“高适应性重组抗体库的构建、筛选及进化技术研究”(2018YFC1602902)的大力支持。
关键词
单链抗体
氨基糖苷类抗生素
药物残留检测
分子识别机制
间接竞争酶联免疫吸附法
Single-chain antibody
Aminoglycosides
Drug residue detection
Molecular recognition mechanism
Indirect competitive enzyme-linked immunosorbent assay
作者简介
王方雨(1978-),男,河南南阳人,副研究员,博士,从事生物技术研究。
