抗恩诺沙星噬菌体单链抗体库的构建及鉴定

Construction and identification of anti-ENR phage display scFv libraries

本研究以兽药恩诺沙星为对象,构建噬菌体抗体库,为低成本、快速制备目的抗体提供新的途径。以恩诺沙星-鸡卵清蛋白(ENR-OVA)为免疫原对Balb/c小鼠进行免疫,取其脾细胞提取总RNA,并分别扩增全套抗体轻、重链基因,通过重叠延伸PCR技术,以编码柔性多肽(Gly3Ser)4的基因为接头,将轻重链基因组装为完整的scFv基因,将之克隆入pCANTAB5E载体,转化大肠杆菌XL1-Blue,以辅助噬菌体M13KO7对其进行超感染,构建噬菌体抗体库并进行富集、筛选和鉴定;构建了库容量约为2.2×106的抗恩诺沙星噬菌体单链抗体库,并筛选出26株阳性克隆,为表达单链抗体、建立免疫检测方法奠定基础。

This study focuses on construction and identification of immunized phage display library from splenocytes of hyperimmunized BALB/C mice for screening and isolation of scFv fragment against ENR,an alternative method to produce antibodies for veterinary drug residues detection.The total RNA was isolated from splenocytes of a BALB/C mouse hyperimmunized with the Enrofloxacin conjugated to chicken OVA.Variable light and heavy domains of the immunoglobulin genes were amplified by PCR and assembled to produce full-length single-chain Fv（scFV） by overlap extension PCR using a linker primer containing flexible polypeptide-（Gly3Ser）4.The scFv DNA fragment was ligated into phagemid vector pCANTAB5E and electroporated into E.coli XL1-Blue cells.The transformed cells were rescued by M13KO7 helper phage and phage libraries were constructed.The size of antibody libraries is 2.2×106.Following the construction of phage display scFv libraries,the recombinant phage displaying scFv were enriched and identified.There are 26 clones against ENR generated in this study.