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构建了抗CD3/抗CD19(Diabody)微型双功能抗体,并且为增强其稳定性进一步改造为二硫键稳定的抗CD3/抗CD19(ds-Diabody)微型双功能抗体,表达并纯化后测定其生物学活性。构建二硫键稳定的抗CD3/抗CD19表达载体p AYZds CD3CD19,转化感受态大肠杆菌16C9进行原核表达,分离纯化后,经12%SDS-PAGE及Westen-blot鉴定。应用间接免疫荧光和竞争性免疫荧光结合流式细胞分析技术(FACS)检测ds-Diabody与CD19+Raji细胞和CD3+Jurkat细胞的结合活性。改造后的ds-Diabody能够在原核表达系统中进行可溶性表达,ds-Diabody在非还原性SDS-PAGE中,在45 k处可见一条带,Westen-blot在同一位置显示有带,在还原性SDSPAGE中45 k处条带消失,在28 k和26 k各有一条带,Western-blot在28 k显示有带,这些均与理论相符,45 k为二硫键稳定的二聚体,在还原剂巯基乙醇作用下二硫键断开,形成28 k和26 k的2条带。FACS检测ds-Diabody可与CD19+Raji细胞和CD3+Jurkat细胞特异结合,并能竞争性抑制单抗HIT3a和HIT19a与上述细胞的结合。说明改造后的二硫键稳定的抗CD3/抗CD19双功能抗体既能在原核系统中进行可溶性表达,同时又保留了与细胞的结合活性。
The anti-CD3 / anti-CD19 (diabody) micro-bifunctional antibody was constructed and further modified to be disulfide-stabilized anti-CD3 / anti-CD19 (ds-Diabody) microbifunctional antibody to enhance its stability. After expression and purification, Biological activity. The disulfide-stabilized anti-CD3 / anti-CD19 expression vector p AYZds CD3CD19 was constructed and transformed into competent E.coli 16C9 for prokaryotic expression. After separation and purification, it was identified by 12% SDS-PAGE and Westen-blot. The binding activity of ds-Diabody to CD19 + Raji cells and CD3 + Jurkat cells was examined by indirect immunofluorescence and competitive immunofluorescence combined with flow cytometry (FACS). The reconstructed ds-Diabody was able to express soluble in prokaryotic expression system. Ds-Diabody showed a band at 45 kDa in non-reducing SDS-PAGE, Westen-blot showed band at the same position, The band at 45 kPa disappeared, one band at 28 k and 26 k, and Western-blot showed bands at 28 k, all in agreement with the theory. The 45 k disulfide-stabilized dimer, Disulfide bonds were broken by mercaptoethanol, forming two bands of 28 k and 26 k. FACS assay ds-Diabody specifically binds to CD19 + Raji cells and CD3 + Jurkat cells and competitively inhibits the binding of mAbs HIT3a and HIT19a to these cells. This shows that the modified disulfide-stabilized anti-CD3 / anti-CD19 bifunctional antibody can both express soluble in prokaryotic system, while retaining the binding activity with cells.