抗脂多糖单链噬菌体抗体在大肠埃希菌中的表达及筛选

Expression and Screening of Anti-lipopolysaccharide Single-chain Phage Antibody in E.coli

目的 研究小鼠鼠源性的抗脂多糖 (lipopolysaccharide,L PS)单链可变区片段 (single- chain fragmentvariable,Sc Fv) ,即在大肠埃希菌 TG1中的表达 ,并筛选出对 L PS具有较高亲和力的 Sc Fv。方法 将预先构建好的有效库容量为 4 .75× 10 6 的噬菌体抗体库接种至 SOBAG培养基上 ,隔夜培养后扩容至约 1× 10 1 0 ;严格控制lac启动子的表达 ,仅当加入 M13KO7辅助噬菌体后才使 Sc Fv以附着型的形式表达 ,然后利用 L PS淘筛特异性的 Sc Fv,富集的噬菌体阳性克隆重新感染 TG1;最后随机挑选出 190个菌落在 96孔培养板分别培养单个含特异性 Sc Fv的 TG1菌落 ,经间接 EL ISA检测抗 L PS的 Sc Fv。结果 淘筛一轮过后即有 3× 10 4 阳性菌落长出 ,190个菌落经间接 EL ISA检测有两个阳性克隆。结论 成功地使鼠抗 L PS Sc Fv在大肠埃希菌中表达 ,并从中筛选出了两株抗 L PS Sc Fv。

OBJECTIVETo search the expression of murine anti-lipopolysaccharide (LPS) single-chain fragment variable (ScFv) phage antibody in Escherichia coli and to screen ScFv that have high affinity to LPS. METHODSThe transformed TG1 bacteria were first incubated in SOBAG medium with 24 hours. The library was rescued with M13KO7 helper phage and the inducible lac promoter was tightly controlled to yield recombinant phage that displays ScFv on its tips. The phage-displayed antibodies capable of binding specific LPS antigen could be seleced by panning against LPS. Then enriched positive clones were reinfected into TG1 cells. Finally, 190 clones were randomly selected and rescued individually to detect the anti-LPS antibodies with indirect ELISA.RESULTS A total of 3×104 colonies were gotten after panning once. Two positive colonies were confirmed with indirect ELISA among 190 randomly selected colonies.CONCLUSIONSMurine anti-LPS ScFv was successfully expressed in E. coli and two anti-LPS ScFvs were successfully screened.