MAP30 (Momordica antiviral protein 30kD) is a single-chain Ⅰ-type ribosome inactivating protein with a variety of biological activities, including anti-tumor ability. It was reported that MAP30 would serve as a novel and relatively safe agent for prophylaxis and treatment of liver cancer. To determine whether adding two tumor targeting peptides could improve the antitumor activities of MAP30, we genetically modified MAP30 with an RGD motif and a EGFRi motif, which is a ligand with high affinity for αvβ3 integrins and with high affinity for EGFR. The recombinant protein ELRL-MAP30 (rELRL-MAP30) containing a GST-tag was expressed in E. coli. The rELRL-MAP30 was highly expressed in the soluble fraction after induction with 0.15 mM IPTG for 20 h at 16 °C. The purified rELRL-MAP30 appeared as a band on SDS–PAGE. It was identified by western blotting. Cytotoxicity of recombinant protein to HepG2, MDA-MB-231, HUVEC and MCF-7 cells was detected by MTT analysis. Half maximal inhibitory concentration (IC₅₀) values were 54.64 μg/mL, 70.13 μg/mL, 146 μg/mL, 466.4 μg/mL, respectively. Proliferation inhibition assays indicated that rELRL-MAP30 could inhibit the growth of Human liver cancer cell HepG2 effectively. We found that rELRL-MAP30 significantly induced apoptosis in liver cancer cells, as evidenced by nuclear staining of DAPI. In addition, rELRL-MAP30 induced apoptosis in human liver cancer HepG2 cells by up-regulation of Bax as well as down-regulation of Bcl-2. Migration of cell line were markedly inhibited by rELRL-MAP30 in a dose-dependent manner compared to the recombinant MAP30 (rMAP30). In summary, the fusion protein displaying extremely potent cytotoxicity might be highly effective for tumor therapy.

MAP30（苦瓜抗病毒蛋白30kD）是一种单链Ⅰ型核糖体失活蛋白，具有多种生物活性，包括抗肿瘤能力。据报道，MAP30 将作为一种新型且相对安全的肝癌预防和治疗药物。为了确定添加两种肿瘤靶向肽是否可以提高 MAP30 的抗肿瘤活性，我们对 MAP30 进行了基因修饰，具有 RGD 基序和 EGFRi 基序，这是一种对 αvβ3 整合素具有高亲和力和对 EGFR 具有高亲和力的配体。含有 GST 标签的重组蛋白 ELRL-MAP30 (rELRL-MAP30) 在大肠杆菌中表达. rELRL-MAP30 在 16 °C 下用 0.15 mM IPTG 诱导 20 小时后在可溶性部分中高度表达。纯化的 rELRL-MAP30 在 SDS-PAGE 上显示为条带。通过蛋白质印迹鉴定。MTT分析检测重组蛋白对HepG2、MDA-MB-231、HUVEC和MCF-7细胞的细胞毒性。半数最大抑制浓度（IC ₅₀) 值分别为 54.64 μg/mL、70.13 μg/mL、146 μg/mL、466.4 μg/mL。增殖抑制试验表明，rELRL-MAP30可以有效抑制人肝癌细胞HepG2的生长。我们发现 rELRL-MAP30 显着诱导肝癌细胞凋亡，DAPI 的核染色证明了这一点。此外，rELRL-MAP30 通过上调 Bax 和下调 Bcl-2 诱导人肝癌 HepG2 细胞凋亡。与重组 MAP30 (rMAP30) 相比，rELRL-MAP30 以剂量依赖性方式显着抑制细胞系的迁移。总之，显示出极强细胞毒性的融合蛋白可能对肿瘤治疗非常有效。