Production and Directional Evolution of Antisarafloxacin ScFv Antibody for Immunoassay of Fluoroquinolones in Milk.
Production and Directional Evolution of Antisarafloxacin ScFv Antibody for Immunoassay of Fluoroquinolones in Milk.
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DOI：
10.1021/acs.jafc.6b03356
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发表时间：
2016-10
影响因子：
6.1
通讯作者：
J. P. Wang;Jun Dong;C. Duan;Hui Zhang;Xin He;G. Wang;G. Zhao;Jing Liu
中科院分区：
文献类型：
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作者：
J. P. Wang;Jun Dong;C. Duan;Hui Zhang;Xin He;G. Wang;G. Zhao;Jing Liu
将其基因直接转化Rosetta-gami（DE 3）表达，获得重组抗沙拉沙星ScFv抗体，并采用分子对接方法研究其对12种氟喹诺酮类药物的识别机制。在虚拟突变的基础上，将单链抗体的接触氨基酸Tyr 99定向突变为His。ScFv突变体显示出对12种药物的亲和力高度增加，灵敏度提高了7倍。最后，利用该突变株建立了牛奶中12种药物的间接竞争酶联免疫吸附测定方法。方法的检出限为0.3-8.0 ng/mL，相关系数为5- 106%，加标回收率为62.0- 89.3%。这是第一个研究报告的进化的单链抗体使用定向诱变策略的基础上虚拟突变。
A recombinant antisarafloxacin ScFv antibody was produced by direct transformation of its gene into Rosetta-gami(DE3) for expression, and then its recognition mechanisms for 12 fluoroquinolones were studied using the molecular docking method. On the basis of the results of virtual mutation, the ScFv antibody was evolved by directional mutagenesis of contact amino acid residue Tyr99 to His. The ScFv mutant showed highly increased affinity for the 12 drugs with up to sevenfold improved sensitivity. Finally, the mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 12 drugs in milk. The limits of detection were in the range of 0.3-8.0 ng/mL; the ties were in the range of 5-106%, and the recoveries from the standard fortified blank milk were in the range of 62.0-89.3%. This is the first study reporting the evolution of an ScFv antibody using a directional mutagenesis strategy based on virtual mutation.