抗Pgp基因工程抗体ScFv的构建、表达及其活性测定

Expression and characterization of recombinant ScFv antibody detecting p-glycoprotein

目的 :构建表达抗PgpScFv,进行体外活性的测定。方法 :利用RT PCR方法 ,克隆抗Pgp杂交瘤细胞PHMAO2的重链可变区基因 (VH) ,轻链可变区基因 (VL) ,拼接为单链抗体 (ScFv) ,再克隆到具有强启动子的PET2 8a(+)载体上进行表达 ,并进行了表达产物体外活性的测定。结果 :构建了表达质粒PET2 8a(+) ScFvPGP。表达产物可与表达Pgp抗原的K5 6 2 A0 2细胞特异性结合 ,并不抑制Pgp外排泵的功能。结论 :构建表达的抗PgpScFv只有针对Pgp抗原的识别功能 ,并无抑制作用 ,因此应用于人体后不会干扰正常细胞的排泄分泌功能 ,无论是作为靶向诊断治疗的载体还是作为双功能抗体的一臂 ,均具有广泛的应用前景。

Objective:Expression and characterization of recombinant ScFv antibody detecting P-glycoprotein.Methods:Amplify the gene of variable region of heavy chain(VH) and light chain (VL) of the antibody against P-glycoprotein from hybridoma cell line PHMA02. VH and VL genes were joined as single chain Fv (ScFv) by a flexible peptide linker and cloned into the plasmid PET28a(+) to construct the ScFv expression vector PET28a(+)-PgpScFv,and expressed from E.coli.Results:The anti-Pgp ScFv was expressed,isolated and purified. Like the native Mab PHMA02,the recombinant ScFv showed specific binding activity for the resistant cell line K562/A02 ,but can not block the activity of Pgp.