The Expression And Biological Role Analysis Of Reorganization Rana Pipiens Onconase (rrONC) In P. Pastoris

Onconase is the current global mainly one of100new drugs and have very strong killerfunction to a wide variety of tumor of body inside and outside because of its nuclease low andcell toxicity strong,, Therefore, the paper constructs pPIC9K-rrONC expression vector. WithGS115pichia pastorios for receptor, extracted engineering strains from restructuring yeast canexpress correctly rrONC. Then the paper study for decompressing bottle fermentationconditions of engineering strains and for separation and purification methods of rrONC andfor rrONC biological function in vitro.This paper in building engineering bacteria was used for the production of strains ofpPIC9K expression vector and successfully build the restructuring expression plasmidpPIC9K-rrONC. The plasmid integrate to pichia pastorios GS115genome by the electricitytransformation, and by the genome PCR screening, MM and MD flat screen, BMGY/BMMYmedium training, sds-page identification steps detection, and finally got the right engineeringstrains of expression rrONC. And then in the wave bottles of condition, to express thequantity and time relationship, it is found that as induced the extension of time this proteinexpression were normal quantity curve and reached a peak in120h.The engineering bacterium with the BMGY/BMMY culture medium in1L the wavebottle is studied of all kinds of fermentation conditions for optimization. The results foundthat in1L the wave bottle engineering bacterium express restructuring leopard frogs enzymeis the best condition of bacteria liquid product100ml, speed200rpm, pH5.5, temperature25℃, methanol concentration1%,0.5%, and the concentration of ammonium sulfate phosphateconcentration100mM.This paper groped for purification rrONC conditions and the results show that usingisoelectric point precipitation, low temperature ethanol precipitation classification andultrafiltration can get about90%purity of purpose product.This paper preliminary inspection of the rrONC biological activities for amnioticmembrane cells WISH and nine kinds of cancer cells in vitro. MTT method to measure theresults found that rrONC ten kinds of cell activity has inhibitory effect and its inhibition andconcentration into positive correlation; Hoechst33258dyeing results show that in0.5*IC50,IC50and2*IC50three different drug concentrations, rrONC can promote the above tenkinds of cell apoptosis of different level. And so as to further make clear rrONC cell toxicity mechanism to lay the foundation.