| Rhizoma Pinelliae is the dried tuber of Pinellia ternata(Thunb)Breit which belongs to a plant of the family Araceae. It is an important Chinese medicine. Rhizoma Pinell- -iae is a widely distributed plant specie, it distribute in most parts of Southeast Asia, China is rich in the resource, Changjiang River Basin is the most. For internal use, R- -hizoma Pinelliae are drying dampness to resolve phlegm, downbearing the adverse f- -low of Qi to stop vomiting, dissipating palpable and nonpalpable masses; for external use, it can be to reduce inflammation. In addition, it can be used as a pesticide. Rhizo- -ma Pinelliae contain many active ingredients, for example, active polysaccharides, alkaloid, sterol, pinellin and so on. Recent research show, pinellin is the main active ingredient of Rhizoma Pinelliae in anti-tumor, anti-fertility and insecticide.The composition of natural medicine is usually very complex, Pinellia ternata (Th- -unb.)Breit. contains proteins more than 10 species. With the in-depth study of pinelli- -n, how to get a lot of pinellin purification of meeting the research requirements whic- -h will become very important. Now, the main methods of isolation are mainly hydro- -phobic interaction chromatography, affinity chromatography, ion exchange chromat- -ography and so on, but these methods all have some shortcomings, so it can not meet the research requirements. On this basis, pinellin is separated by gel filtration from 0.45 ammonium sulfate saturation of total protein extracted from Pinellia ternata, it is only 80% purity, and its relative molecular weight is about 12KDa. Another pinellin is separated by Sephadex G-50 and MBP Trap HP affinity column from 0.45～0.8 amm- -onium sulfate saturation of total protein extracted from Pinellia ternata, its relative molecular weight is about 40KDa, about 90% purity, but both are very low reproduci- -bility, which is not conducive to follow-up study. Then, pinellin is also separated by hydrophobic chromatography from 0.45 ammonium sulfate saturation of total protein salt out from Pinellia ternata. Although improved reproducibility, 90% purity can be achieved, but the method of demanding operating conditions. Finally, we take advant- -age of the lectin which have a specific affinity to mannose, and use a mannose-Seph- -rose 4B affinity gel to get a mannose-bonding pinellia ternata lectin from the total pr- -oteins of Pinellia ternata which are in the 0.95 ammoniun sulphate solution. The ob- -serve molecular weight is about 12KDa, and the mass spectra assay show that the molecular weight is 12.165KDa after the isolation of gel filtration, and the purity is over 95%. The result of iodic acid-Schiff reagent assay demonstrate that the pinellia ternata lectin is glycoprotein. The result of cruor assay indicate that the lowest conce- -ntration of the blood congealed reaction is 25μg/ml, and there is no obvious reaction if the concentration is lower than 12.5μg/ml. Using the amino acid automatic analyzer to analysis the compose of pinellia ternata lectin, the result show that there are 15 species of amino acid, the highest is aspartic acid, the lowest is cysteine.This paper also research that the expression of Pinellia ternata agglutinin(PTA) and the purification conditions of the recombination protein. The main content is that exp- -ression vector pET-28a(+) which contain Pinellia ternata agglutinin(PTA) gene trans- -form into E.coli BL21(DE3). It would be induced by IPTG. Then SDS-PAGE analys- -is show that, there is consistent with the expected protein, the molecular weight is ab- -out 30KDa. The fusion protein exists as the form of inclusion bodies. Separate the inclusion bodies with the method of ultrasonic separation. When the inclusion bodies were dissolved, perform the Ni affinity column purification with the histidine-tagged of proteins.Then select the conditions of purification filter, to obtain the most Pinellia ternata agglutinin of purified expression. |
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