抗GPC3单链抗体细菌外膜囊泡的制备及其肝细胞癌靶向性的鉴定

Preparation of bacterial outer membrane vesicles with anti-GPC3 single-chain antibody and identification of their targeting effects on hepatocellular carcinoma

摘要:

本研究旨在制备抗磷脂酰肌醇蛋白聚糖3 (glypican-3, GPC3)单链抗体的细菌外膜囊泡(outer membrane vesicles, OMVs)，分析其在体内外对Hep G2肝癌细胞及组织的靶向效果。通过合成Hbp-hGC 33-scFv基因并连接至pET28a表达载体，构建重组表达质粒pET28a-Hbp-hGC 33-scFv；采用免疫印迹法检测原核表达的融合蛋白Hbp-hGC 33-scFv，确定其最适诱导条件；采用超滤浓缩法收集重组表达菌株分泌的外膜囊泡(Hbp-hGC 33-OMVs)并进行表征；采用免疫电镜技术分析Hbp-hGC 33-scFv在细菌及Hbp-hGC 33-OMVs的定位情况；采用免疫荧光法观察其与Hep G2细胞的结合效果。建立Hep G2荷瘤小鼠模型，通过活体荧光成像系统观察Hbp-hGC 33-OMVs在小鼠肿瘤部位的靶向滞留情况。结果显示，融合蛋白的实际分子量为175.3 kDa，最佳诱导条件为在OD₆₀₀=0.5 时加入0.5 mmol/L IPTG于16 ℃低温诱导过夜。Hbp-hGC 33-OMVs平均粒径为(112.3±4.6) nm，具有不规则球形结构，表达外膜囊泡标记蛋白OmpC、OmpA和融合蛋白Hbp-hGC 33-scFv。与野生型wtOMVs相比，Hbp-hGC 33-OMVs组对Hep G2细胞的结合效果更强(P=0.008)。活体成像显示，Hbp-hGC 33-OMVs组能快速精准地靶向肿瘤部位。抗GPC3单链抗体细菌外膜囊泡的成功制备为肝细胞癌的特异性靶向治疗奠定了实验基础。

Abstract:

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti- glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD₆₀₀=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.