重组谷糠源过氧化物酶的克隆、表达及逆转结肠癌化疗耐药活性

本课题组前期首次从谷糠中获得具抗肿瘤活性的过氧化物酶FMBP。为了该蛋白质后期在体外的规模化生产，本研究以谷子cDNA为模板，利用基因工程手段构建了pMal-s-FMBP融合质粒，并优化诱导条件进行原核表达与纯化，最终获得纯度大于90 %的具抗肿瘤活性的重组FMBP（Re-FMBP）。研究发现，Re-FMBP可以逆转HCT-8/5-Fu耐药细胞对5-Fu的耐药性，耐药逆转倍数达到9.6倍。通过Annexin V/碘化丙啶双染色流式细胞仪检测显示：Re-FMBP能够诱导HCT-8/5-Fu耐药细胞发生凋亡；通过罗丹明123染色，流式细胞仪检测结果显示，Re-FMBP处理后，HCT-8/5-Fu耐药细胞内的荧光强度增强。说明Re-FMBP能够增加5-Fu药物在HCT-8/5-Fu耐药细胞内的蓄积。结果揭示，Re-FMBP蛋白可通过抑制耐药细胞增殖，诱导耐药细胞凋亡，抑制耐药细胞对5-Fu药物外排功能来增加HCT-8/5-Fu耐药细胞对5-Fu药物的敏感性。因此，谷糠来源的Re-FMBP蛋白可以作为肿瘤化疗辅助药物来开发。

Cloning,Expression and Reversal of Chemotherapeutic Activity of Recombinant Peroxidase from Foxtail Millet Bran

FMBP, a novel peroxidase protein with antitumor activity found in previous study, was extracted and purified from foxtail millet bran. In order to produce large quantities of FMBP in vitro, the pMal-s-FMBP fusion plasmid was constructed with the cDNA of foxtail millet as a template by the genetic engineering method, and then a recombinant FMBP (Re-FMBP) with a purity greater than 90% was obtained through optimizing the induction expression conditions in prokaryotic. MTT assays revealed that Re-FMBP had significant anti-tumor activity. The study found that Re-FMBP could significantly reverse the resistance of HCT-8/5-Fu cells to 5-Fu by 9.6 fold. Flow cytometry analysis showed that Re-FMBP could significantly induce apoptosis in HCT-8/5-Fu cells. The fluorescence intensity of rhodamine 123 was increased in the HCT-8/5-Fu cells stained with rhodamine 123 in a concentration-dependent manner, which indicated that Re-FMBP remarkably increased the accumulation of chemotherapy drugs in HCT-8/5-Fu cells. The present data implied that Re-FMBP significantly enhanced the sensitivity of chemotherapeutic drugs through inhibiting cell proliferation, promoting cell apoptosis and increasing the accumulation of rhodamine-123 (Rh-123) in HCT-8/5-Fu cells. Collectively, these results indicated that Re-FMBP could be potentially used as a new drug-resistance reversal agent in colorectal carcinoma.