| There are three categories of toxin produced by Amanita phalloides according to their structures and properties: amatoxins (AT), phallotoxins (PT) and virotoxins (VT). They are all the small cyclic molecules with steady chemical properties. They can be applied widely in many research fields, such as specifically inhibition of mRNA synthesis in eukaryote, localization of genes in a variety of cells and tissues, gene expression and its control, identification of cell membrane transport system, studying on cell structure and function, and research on tumor and virus. However, toadstools resources are limited and the active compounds are very difficult purified. Thus, all corresponding toxic polypeptide products are imported from European countries and the Unite State. This situation makes against our domestic study and the application of toxic polypeptides.The compond a-amanitin from Amanita virosa was separated and determined by reversed-phase high performance liquid chromatography and UV-spectrum, by using 0.02mol/L aqueous ammonium acetate-acetonitrile as mobile phase with gradient elution mode. The retention time of a-amanitin was about 12.22min. It could be purified from the H2O extraction of Amanita virosa, and its content reached about 2838.8ug in every gramme dry fruiting bodies. Others peptide toxins of Amanita fungi could also be isolated and purified by this method.Zanotti G. attempted to perform the synthesis of amaninamide, a kind of amatoxin. Synthesizing liner octapeptide carbon skeleton was the first step in the process of producing biological - active amatoxin, but it is of high cost andtime-consuming. Since prokaryotes are not sensitive to amatoxins, we planed to construct and express the fusion gene including the coding sequence of octapeptide to produce carbon skeleton, then to circulize the linear peptide circular for biological active polypeptides and to provide a precuror for further derivatization. We used adding-PCR to construct and express fusion gene of linear octapeptide carbon skeleton of amatoxins and glutathione-S- transferase (GST) for the further study. The products of two rounds adding-PCR were connected to the expression vector pGEX-RT. The recombinant including octapeptide carbon skeleton of amatoxins. named pGAT-1, was selected. The sequencing result demonstrated that the fusion gene GST-AT had right phase and open reading frame. The amount of the fusion protein was increased by 33.5% under the optimized condition. This would provide the source of the carbon skeleton for the study of cyclization of linear a-amanitin and active a-amanitin in cultured toadstools.We injected a-amanitin injection into abdominal cavity and caudal vein by two administration manner (dosage: 0.6~0.9 mg/kg and 0.25~0.45mg/kg, respectively), and most mice died after 24~60h of administration. The probit transformation of death rate and the logarithmic transformation of a-amanitin concentration were correlated and regressed. The linear regression equation was Y = 0.0404.Y -0.1297 (R2=0.9662) and Y = 0.0933X-0.4878 (R2=0.9625), respectively. Medial lethal dose (LD50) was 0.742mg/kg and 0.327mg/kg, respectively.The responsive time and intensity after injection of a-amanitin in caudal vein are more serious than that in abdominal cavity, which demonstrated the intoxication symptom and death rate were relevant to the high concentration of drug in blood, a-amanitin intoxication would cause the severe gastroenteritis and rapid descent of body weight in mice. After 12~96h of injection in caudal vein, WBC, RBC and HGB also decreased significantly. The change tendency of micro-cells of WBC was similar to that of leukocytes, while that of macro-cells of WBC significantly reduced after 12h, whose proportion of total leukocytes decreased continually. BUN and Crea in serum increased significantly after 12h. After 24h, the content of ALT and AST was 1308.5± 109.81U/L and 898.0 ± 197.99U/L, respectively, which increased 24.0and 9.64 times, respectively. The content of TBIL and DBIL was 24.21 ± 8.49umol/L and...
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