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  • Prašičji imunski interferon : magistrsko delo = Porcine immune interferon : master thesis
    Cencič, Avrelija
    In present work we describe a model of separation of leukocytes' fractions that are used for production of natural porcine interferon gamma, using asymmetric celluloseacetate (CA) membranes with ... defined porosity. CA-membraneswere prepared through phase-inversion process of thin layer solution of polymer with defferent concentrations of CA(14.8% - 22%) in propanone/water. The nominal thickness of the membranes varied from 0.25 - 0.30 mm. Prepared membranes were characterized with deionised water flux in Amicon stirred cell, under different pressures (2-4 bars). CA1- and CA2-membranes (0.25 mm nominal thickness) were used for the separation of the leukocytes fractions (microfiltration). CA5-membrane (20% concentration of CA in the initial solution of polymer) was found to be the most appropriate for the ultrafiltration (purificatin of natural interferon gamma). The obtained natural porcine interferon gamma, has the specific activity of 1.5 X 10 6 U per mg protein. SDS-page electrophoresis profile showed two IFN species with molecular masses of monomers 15.000 and 20.000. The difference is probably dueto the number of truncated aminoacids and rate of glycosylation. Antiproliferative activity of both IFN species was relatively high, tested on HeLa and REF cell lines. Suggested system of separation of blood cells and production of natural porcine interferon has at least the same efficiency as standard methods and many advantages (lower cost, biocompatibility, non-toxicity)(20,69). It could be applied to the human system of blood separation as well. In parallel we constructed the plasmid containing MetPoIFN-gama cDNA sequence using pET14b and pET22b(+) plasmid vectors, that allowed expression of mature recombinant porcine interferon gamma in the cytoplasm of bacteria. Plasmid vector containing MetPoIFN-gama insert, was then transformed to E.coli BL21 (DE3) strain. Produced recombinant porcine interferon gamma was found soluble and accumulated in cytoplasm inclusion bodies in relatively large amounts (4.5 - 7 mg per g of bacteria's wet pellet). We purifield soluble and inclusion bodies associated recombinant porcine interferon using an ion-exchange cromatography (CM-sepharose Cl-6B) under very mild conditions that allowed high rate of purification and low rateof denaturation. For purification of inclusion bodies we found 8M urea sufficient. Also better refolding was achieved using the mentioned procedure in comparison with guanidine chloride commonly used. Among the bacteria's proteases inhibitors, fetal calf serum (1%) gave the best results (the amount of 15.000 rPoIFN-gama species is significantly lower than with use of PMSF). The protocol we suggested was effective, giving almost pure recombinant porcine interferon gamma with specific activity of 2 X 10 6 U per mg of protein. In comparison betwen natural and recombinant porcine interferon gammathere is no difference in the terms of pH stability. They are considered stable in acidic pH (2 and 2.5) and much less stable at pH 11, according to the antiviral activity measured on MDBK cells, using VSV as a challenger virus. Antiproliferative activity of recombinant porcine interferon gamma is significantly lower in comparison with its natural counterpart.
    Vrsta gradiva - magistrsko delo
    Založništvo in izdelava - Ljubljana : [A. Cencič], 1995
    Jezik - slovenski
    COBISS.SI-ID - 176463

Knjižnica Signatura – lokacija, inventarna št. ... Status izvoda
Fakulteta za kmetijstvo in biosistemske vede, Maribor Čitalnica
m 579 CENCIČ, A. Prašičji
IN: 19950369 		prosto - za čitalnico
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