Lsolation And Purification Of Ribosome-inactivating Proteins(RIPs) From Bitter Melon Seeds And Activity Assay Of α-MMC,β-MMC And Immunotoxin Containing β-MMC

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.