重组靶向核糖体失活蛋白luffin-α-NGR的制备及抗肿瘤活性评价

Preparation of a recombinant tumor-targeting ribosome inactivating protein luffin-α-NGR and evaluation of its antitumor activity

摘要:

将丝瓜籽核糖体失活蛋白luffin-α与肿瘤靶向肽NGR融合，制备luffin-α-NGR重组蛋白，并检测其对肿瘤细胞与血管生成的抑制活性。通过引物设计及PCR扩增获得luffin-α-NGR融合基因，与pGEX-6p-1载体连接后获得pGEX-6p-1/luffin-α-NGR重组质粒，质粒转入大肠杆菌(Escherichia coli) BL21中表达，以GST亲和层析法分离纯化目的蛋白。MTT比色法、Transwell迁移实验以及鸡胚尿囊膜实验(CAM)检测其活性。结果表明，成功获得全长为849 bp的luffin-α-NGR融合基因，目标蛋白在16℃、0.5 mmol/L IPTG诱导16 h后获得最佳表达，经SDS-PAGE和Western blotting分析，GST融合蛋白分子量为56.6 kDa，与预期一致。重组蛋白经GST亲和层析、蛋白酶精准酶切去除标签后，利用MTT法验证其对HepG2和MDA-MB-231细胞的抑制效果显著优于luffin-α。Transwell迁移实验和CAM实验证实重组蛋白luffin-α-NGR对肿瘤细胞迁移和血管生成具有明显的抑制作用。本研究成功地制备了重组蛋白luffin-α-NGR，并证实该重组蛋白对肿瘤细胞具有良好的抑制活性，为后续重组靶向蛋白药物的开发奠定基础。

Abstract:

Loofah seeds ribosome inactivating protein luffin-α was fused with a tumor-targeting peptide NGR to create a recombinant protein,and its inhibitory activity on tumor cells and angiogenesis were assessed.luffin-α-NGR fusion gene was obtained by PCR amplification.The fusion gene was ligated with pGEX-6p-1 vector to create a recombinant plasmid pGEX-6p-1/luffin-α-NGR.The plasmid was transformed into E.coli BL21,and the target protein was isolated and purified by GST affinity chromatography.The luffin-α-NGR fusion gene with a full length of 849 bp was successfully obtained,and the optimal soluble expression of the target protein was achieved under the conditions of 16℃,0.5 mmol/L IPTG after 16 h induction.SDS-PAGE and Western blotting confirmed the recombinant protein has an expected molecular weight of 56.6 kDa.Subsequently,the recombinant protein was de-tagged by precision protease digestion.The inhibitory effects of the recombinant protein on liver tumor cells HepG2 and breast cancer cells MDA-MB-231 were significantly stronger than that of luffin-α.The Transwell and CAM experiment proved that the recombinant protein luffin-α-NGR also had a significant inhibitory effect on tumor cells migration and neovascularization.The inhibitory activity on tumor cells and angiogenesis of the recombinant luffin-α-NGR protein lays a foundation for the development of subsequent recombinant tumor-targeting drugs.