链间二硫键增强抗前胃泌素释放肽单链抗体稳定性

前胃泌素释放肽(pro-gastrin-releasing peptide, ProGRP)是小细胞肺癌的特异性标志物,¹³¹I 标记的抗ProGRP单克隆抗体对小细胞肺癌具有明显的抑制作用。抗ProGRP单链抗体的制备具有重要的应用前景。本研究以抗ProGRP单克隆细胞株E-B₅ cDNA为模板,扩增获得VH和VL序列,并对其进行序列比对和同源建模,分析引入链间二硫键的突变位点。通过基因合成获得单链抗体Scfv_ProGRP 和单链二硫键稳定抗体Sdsfv_ProGRP基因,并将其分别构建在质粒pET-28a,获得重组表达质粒pET28a-His-Scfv_ProGRP 和 pET28a-His-Sdsfv_ProGRP。重组表达质粒转化大肠杆菌BL21(DE3),诱导表达出的ScFv_ProGRP 和SdsFv_ProGRP以包涵体的形式存在。包涵体经变复性后进行纯化,获得ScFv_ProGRP 和SdsFv_ProGRP的纯度分别为87.38%和95.73%。酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测显示,0.021 mg/mL ScFv_ProGRP和0.026 mg/mL SdsFv_ProGRP可以与抗原ProGRP特异性结合。SdsFv_ProGRP稳定性高于ScFv_ProGRP。结果表明,重组表达的抗ProGRP单链抗体识别ProGRP,具有免疫学活性, 链间二硫键增强了抗前胃泌素释放肽单链抗体稳定性,研究为ProGRP人源化单链抗体的制备提供了新的数据。

Interdomain Disulfide Bond Enhances the Stability of Single-chain Antibody Against Pro-gastrin-releasing Peptide

Pro-gastrin-releasing peptide (ProGRP) is a specific marker of small cell lung cancer.¹³¹I-labeled anti-ProGRP monoclonal antibody inhibits proliferation of small cell lung cancer. The preparation of ProGRP single-chain antibody is valuable for its application. In this study, variable region of heavy chain (VH) and variable region of light chain (VL) sequences were amplified from anti-ProGRP monoclonal cell line E-B₅ by RT-PCR. The sequence alignment and homology modeling of the anti-ProGRP single-chain antibody (ScFv_ProGRP) were carried out, and the mutation sites were introduced to form the interdomain disulfide bonds. The genes encoding ScFv_ProGRP and SdsFv_ProGRP containing interdomain disulfide bond were synthesized and cloned into plasmid pET-28a. The pET28a-His-Scfv_ProGRP and pET28a-His-Sdsfv_ProGRP were transformed into E.coli BL21(DE3). ScFv_ProGRP and SdsFv_ProGRP were expressed in the form of inclusion bodies. The inclusion bodies were purified after denaturation and renaturation, and the purity of ScFv_ProGRP and SdsFv_ProGRP were 87.38% and 95.73%, respectively. ELISA showed that 0.021 mg/ mL ScFv_ProGRP and 0.026 mg/ mL SdsFv_ProGRP reacted specifically with ProGRP. Furthermore, SdsFv_ProGRP showed higher stability than ScFv_ProGRP. The results showed that the recombinant anti-ProGRP single-chain antibody reacted with ProGRP and had immunological activity.The interdomain disulfide bond enhanced the stability of ProGRP single-chain antibody. The study provided new data for the preparation of ProGRP humanized single-chain antibody.