Radix Anti-fungal Protein Chromatographic Separation With Mass Spectrometry Identification

This paper included the historical recordation and modern research about Isatis Root. They are the chemistry component, pharmacological function,clinic application, general of big molecules about Isatis Root and the antifungal activity of the traditional Chinese medicine etc. In addition,the text introduced the application of proteomics, protein research technology, application in medical and traditional medical fields. In the experimental part, two hundred of fresh hurbs were screened for antifungal activity against Trichodermaviride and several ones showed antifungal activity. As a result, we discovered Isatis Root showed the most remarkable antifungal activity,furthermore,its active component was proved to be a kind of protein. In succession, begin to purify the active protein. First, prepare the protein extraction buffer to extract the total protein of Isatis Root. Then marinated the grinding Isatis Root tissue with the buffer, centrifuged and purified with the method of saturated (NH4)2SO4 precipitation. We got four parts of precipitation.Tested their antifungal activity and went on Liquid chromatographies purification with the parts which had remarkable activity. Liquid chromatography purification concluded once cation-exchange(SP-Sepharose Fast Flow, 26/30),twice cation-exchange (Mono S, 1mL),once hydrophobic interaction chromatography (Rersouce ISO, 1mL). During each steps of Liquid chromatography purification, we tested the antifungal activity of each chromatography peak and went on with the active part. At last, we got three active purified protein. They have remarkable antifungal activity against Trichodermaviride,Gaeumannomyces graminis,Fusarium graminearum, Botryosphaeria ribis,Magnaporthe grisea,Pythium spp,Phomopsis asparagi Sacc Bubak, Fusarium oxysporum f.sp,etc. The three pure antifungal proteins showed three clear bands on SDS-PAGE. MALDI-TOF showed that the molecular of the three protein mass was 26642D,31968Dand 22307 D. Then, we picked proteins spot from the SDS-PAGE gel usingthe Ettan spot picker. After digestion with trypsin,the proteins changedto peptides. MALDI-TOF showed their peptide Mass spectrum. On internet,compared them to the data-base and we discovered that they were threenew proteins. With the development of the society, Fungal infections becomemore and more serious problems during the treatment of immunosuppressiveviral infections, malignant tumors, and other immunocompromisingdisorders. Novel drugs with potent antifungal activities have beendeveloped, but they have insufficiently controlled fungal infectionsin immunocompromised patients, and a new strategy for control is desiredThe mechanism of the antifungal protein or peptide is different fromthe usual antibiotics. They interfered the superficial structure ofthe fungal cell and destroyed it. Furthermore, to them, drug-resistance are not easy to emerge.These kinds of drugs are hopeful.So this research broadened our idea for discovering new antifungalmedicine.