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投稿时间：2019-01-29
投稿时间：2019-01-29
中文摘要: 为实现甲硝唑单链抗体（single-chain variable fragment，scFv）在大肠杆菌中的可溶性表达，以甲硝唑抗体可变区基因（VH、VL）为模板，扩增甲硝唑的scFv 基因，scFv 双酶切后与PLIP6/GN 载体重组，构建重组质粒PLIP6/GN-scFv，转化大肠杆菌JM109，阳性克隆转大肠杆菌BL21 经温度诱导表达重组单链抗体，并通过聚丙酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE）对其鉴定。采用竞争酶联免疫（enzyme linked immunosorbent assay，ELISA）检测重组单链抗体的抗原结合活性。结果表明诱导表达的scFv 分子量接近33 kDa，能够特异性识别兽药甲硝唑，半抑制率（IC50）为（0.72±0.04）ng/mL。选择虾样品进行添加回收试验，建立的方法检测其回收率在88.54%到113.27%之间。这说明成功构建重组质粒PLIP6/GN-scFv 并实现可溶性表达并可用于实际样品检测。
Abstract:To obtain soluble expression of anti-metronidazole single-chain variable fragment（scFv）in Escherichia coli，variable region genes （VH，VL）were as a template and amplified for scFv genes，then scFv were double digested and recombined with PLIP6/GN plasmid.Finally，The recombinant vector were transformed into E.coli JM109 that was temperature-induced and expressed recombinant protein.Recombinant protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）and indirected competitive enzyme linked immunosorbent assay （ELISA）.The results showed that the expression of scFv was 33 kDa，and could competitively recognized with metronidazole，the 50%inhibitory concentration（IC50）was（0.72±0.04）ng/mL.Recovery test was proceeded using shrimp sample，the recovery results were ranged from 88.54%to 113.27%based on developed method.This study suggested that the recombinant plasmid PLIP6/GN-scFv was constructed and expresses the scFv gene of metronidazole in E.coli successfully.
文章编号：201909008 中图分类号： 文献标志码：
基金项目:河北省高等学校科学技术研究项目（QN2016242）
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