Department of Pathology, 920th Hospital of the Joint Logistics Support Force of PLA, Kunming, Yunnan, People's Republic of China.
The Graduate School, Kunming Medical University, Kunming, Yunnan, People's Republic of China.
PLoS One. 2022 Jun 1;17(6):e0269084. doi: 10.1371/journal.pone.0269084. eCollection 2022.
Cell membrane penetrating peptide BR2 can bind with ganglioside and introduce foreign drugs into tumor cells. In this study, we employed BR2 to carry the broad-spectrum anti-p21Ras scFv prepared in our laboratory into ganglioside expressing tumor cells for therapy of ras-driven tumors.
BR2-p21Ras scFv gene was cloned to prokaryotic expression vector and expressed in E. coli BL21, then the fusion protein was purified with HisPur Ni-NTA. The immunoreactivity of the fusion protein with p21Ras was detected by ELISA and western blotting. The membrane-penetrating and immune co-localization with p21Ras of the fusion protein were determined by immunofluorescence. The antitumor activity was investigated using MTT, wound healing, colone formation, and apoptosis assays in vitro.
BR2-p21Ras scFv fusion protein was successfully expressed and purified. We found that the fusion protein could specifically penetrate into human tumor cell lines which express ganglioside including human neuroblastoma cell line SK-N-SH, human colon cancer cell line HCT116 and human glioma cell line U251. After entering tumor cells the fusion protein bonded specifically with p21Ras. In vitro experiments revealed that it could significantly inhibit the proliferation, migration, and colone formation of HCT116, SK-N-SH, and U251 cells and promote the apoptosis of these tumor cells.
BR2-p21Ras scFv can penetrate ganglioside expressing tumor cells and inhibit the growth of ras-driven tumor by binding with p21Ras, and producing an inhibitory effect. It is suggested that BR2-p21Ras scFv is a potential ras-driven tumor therapeutic antibody.
细胞膜穿透肽 BR2 可与神经节苷脂结合，并将外源性药物导入肿瘤细胞。在本研究中，我们采用 BR2 将本实验室制备的广谱抗 p21Ras scFv 导入表达神经节苷脂的肿瘤细胞，用于治疗 ras 驱动的肿瘤。
将 BR2-p21Ras scFv 基因克隆到原核表达载体中，并在大肠杆菌 BL21 中表达，然后用 HisPur Ni-NTA 纯化融合蛋白。通过 ELISA 和 Western blot 检测融合蛋白与 p21Ras 的免疫反应性。通过免疫荧光法测定融合蛋白的膜穿透性和与 p21Ras 的免疫共定位。通过 MTT、划痕愈合、集落形成和凋亡实验在体外研究其抗肿瘤活性。
成功表达和纯化了 BR2-p21Ras scFv 融合蛋白。我们发现融合蛋白可以特异性穿透表达神经节苷脂的人肿瘤细胞系，包括人神经母细胞瘤细胞系 SK-N-SH、人结肠癌细胞系 HCT116 和人胶质瘤细胞系 U251。进入肿瘤细胞后，融合蛋白与 p21Ras 特异性结合。体外实验表明，它可以显著抑制 HCT116、SK-N-SH 和 U251 细胞的增殖、迁移和集落形成，并促进这些肿瘤细胞的凋亡。
BR2-p21Ras scFv 可以通过与 p21Ras 结合穿透表达神经节苷脂的肿瘤细胞，并抑制 ras 驱动的肿瘤生长，产生抑制作用。提示 BR2-p21Ras scFv 是一种有潜力的 ras 驱动肿瘤治疗抗体。
