苦瓜籽核糖体失活蛋白的分离纯化及对细胞抑制活性的研究
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摘要
研究人员已从苦瓜籽中分离出包括 α-Momorcharin (α－MMC)、β－MMC、γ－MMC、δ－MMC、ε－MMC和MAP30等6种苦瓜素。苦瓜素具有抑制兔网织红细胞裂解液中蛋白质合成的活性，其半数抑制浓度IC50为0.01～10.0nM。其中，α－MMC和β－MMC的抑制活性强，其IC50分别为0.23nM和0.19nM，且在苦瓜籽中的含量丰富。
本研究利用α－MMC和β－MMC的高等电点特性，首先在色谱工作站上让苦瓜籽粗提液流过弱阴离子DEAE-650C与弱阳离子CM-650M串联交换柱，然后对CM-650M柱进行低梯度缓慢洗脱，得到纯化的α－MMC和β－MMC；发现α－MMC对香蕉枯萎菌和果腐霉菌、β－MMC对果腐霉菌均具有较强的抑制活性；研究了α－MMC和β－MMC对细胞的抑制活性，结果表明α－MMC和β－MMC对人肝癌细胞Bel-7402的半数抑制浓度IC50分别为0.073mg/mL和0.033mg/mL，对人脐静脉内皮细胞Hunec的半数抑制浓度IC50分别为 0.074mg/mL和0.036mg/mL，可知β－MMC对细胞的抑制活性要比α－MMC强；给β－MMC偶联上抗肝癌单克隆抗体HAb18F（ab’）2后，生成免疫毒素(～HAb18F（ab’）2，其对Bel-7402的半数抑制浓度IC50为1.21×10-06mol/L，比β-MMC对Bel-7402的半数抑制浓度1.10×10-06mol/L略高；免疫毒素对Hunec的半数抑制浓度IC50为7.42×10-05mol/L，是β－MMC对Hunec的半数抑制浓度1.20×10-06mol/L的61.8倍，表明β-MMC偶联上抗肝癌单克隆抗体片段HAb18F(ab’)2后具有靶向作用的功能。
本研究探讨了利用苦瓜籽核糖体失活蛋白β－MMC来研制靶向抗肿瘤药物的可能性，同时也为用阴阳离子交换串联色谱分离纯化碱性或酸性蛋白探索了一条新的技术路线。
Researchers had purified out α－MMC、β－MMC、γ－MMC、δ－MMC、ε－MMC and MAP30 from bitter melon seeds and these RIPs showed out inhibitory activities of protein synthesis in rabbit reticulocyte lysate and their IC50 was from 0.01nM to 10nM. IC50 of α－MMC and β－MMC was 0.23nM and 0.19nM among them. This illusminated α－MMC and β－MMC had high inhibitory activity. We connected anion exchange chromatograpy column (DEAE-650C) and cation exchange chromatography column (CM-650M) in series on perfusion charomatography workstation to isolate and purify α－MMC and β－MMC on the base of their high pI. Our wanted proteins penetrated out of DEAE-650C firstly and eluted out of CM-650M through slow salt grads. α－MMC and β－MMC inhibited some bacteria, such as Microzyme and Staph.aureus. We researched α－MMC and β－MMC’s antifungal activities on Fusarium oxysporum 、Pythium aphanidermatum and Sclerotium rolfsii and experiments showed they had antifungal activity against Fusarium oxysporum and Pythium aphanidermatum but not against Sclerotium rolfsii. We also researched α－MMC and β－MMC’s cell toxicity. IC50 of α－MMC and β－MMC against Bel-7402 was 0.073mg/mL and 0.033mg/mL and IC50 of α－MMC and β－MMC against Hunec was 0.074mg/mL and 0.036mg/mL.We happed anti-liver cancer monoclone antibody HAb18F(ab’)2 to β－MMC and gained immunotoxin (-HAb18F(ab’)2. IC50 of immunotoxin against Bel-7402 was 1.21×10-06mol/L, a little higher than that of β－MMC; IC50 of immunotoxin against Hunec was 7.42×10-05mol/L, as higher as 61.8 times than that of β－MMC. Experiments showed immunotoxin had the ability of targeting therapy to some specific cells.
Our research developed a good technologic way to isolate and purify basic protein or acid protein ,we also researched the possibility of using ribosme-inactiviting protein β-MMC to make anticancer drug.
本研究利用α－MMC和β－MMC的高等电点特性，首先在色谱工作站上让苦瓜籽粗提液流过弱阴离子DEAE-650C与弱阳离子CM-650M串联交换柱，然后对CM-650M柱进行低梯度缓慢洗脱，得到纯化的α－MMC和β－MMC；发现α－MMC对香蕉枯萎菌和果腐霉菌、β－MMC对果腐霉菌均具有较强的抑制活性；研究了α－MMC和β－MMC对细胞的抑制活性，结果表明α－MMC和β－MMC对人肝癌细胞Bel-7402的半数抑制浓度IC50分别为0.073mg/mL和0.033mg/mL，对人脐静脉内皮细胞Hunec的半数抑制浓度IC50分别为 0.074mg/mL和0.036mg/mL，可知β－MMC对细胞的抑制活性要比α－MMC强；给β－MMC偶联上抗肝癌单克隆抗体HAb18F（ab’）2后，生成免疫毒素(～HAb18F（ab’）2，其对Bel-7402的半数抑制浓度IC50为1.21×10-06mol/L，比β-MMC对Bel-7402的半数抑制浓度1.10×10-06mol/L略高；免疫毒素对Hunec的半数抑制浓度IC50为7.42×10-05mol/L，是β－MMC对Hunec的半数抑制浓度1.20×10-06mol/L的61.8倍，表明β-MMC偶联上抗肝癌单克隆抗体片段HAb18F(ab’)2后具有靶向作用的功能。
本研究探讨了利用苦瓜籽核糖体失活蛋白β－MMC来研制靶向抗肿瘤药物的可能性，同时也为用阴阳离子交换串联色谱分离纯化碱性或酸性蛋白探索了一条新的技术路线。
Researchers had purified out α－MMC、β－MMC、γ－MMC、δ－MMC、ε－MMC and MAP30 from bitter melon seeds and these RIPs showed out inhibitory activities of protein synthesis in rabbit reticulocyte lysate and their IC50 was from 0.01nM to 10nM. IC50 of α－MMC and β－MMC was 0.23nM and 0.19nM among them. This illusminated α－MMC and β－MMC had high inhibitory activity. We connected anion exchange chromatograpy column (DEAE-650C) and cation exchange chromatography column (CM-650M) in series on perfusion charomatography workstation to isolate and purify α－MMC and β－MMC on the base of their high pI. Our wanted proteins penetrated out of DEAE-650C firstly and eluted out of CM-650M through slow salt grads. α－MMC and β－MMC inhibited some bacteria, such as Microzyme and Staph.aureus. We researched α－MMC and β－MMC’s antifungal activities on Fusarium oxysporum 、Pythium aphanidermatum and Sclerotium rolfsii and experiments showed they had antifungal activity against Fusarium oxysporum and Pythium aphanidermatum but not against Sclerotium rolfsii. We also researched α－MMC and β－MMC’s cell toxicity. IC50 of α－MMC and β－MMC against Bel-7402 was 0.073mg/mL and 0.033mg/mL and IC50 of α－MMC and β－MMC against Hunec was 0.074mg/mL and 0.036mg/mL.We happed anti-liver cancer monoclone antibody HAb18F(ab’)2 to β－MMC and gained immunotoxin (-HAb18F(ab’)2. IC50 of immunotoxin against Bel-7402 was 1.21×10-06mol/L, a little higher than that of β－MMC; IC50 of immunotoxin against Hunec was 7.42×10-05mol/L, as higher as 61.8 times than that of β－MMC. Experiments showed immunotoxin had the ability of targeting therapy to some specific cells.
Our research developed a good technologic way to isolate and purify basic protein or acid protein ,we also researched the possibility of using ribosme-inactiviting protein β-MMC to make anticancer drug.
引文
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