Recombinant Pseudomonas Aeruginosa Exotoxin A Series Of Toxin Adp-ribosylation Transferase Activity Analysis

(rPEA) is the single chain fusion protein .Molecular of rPEA is small,molecularstructure is simple, molecular strucflire modification is easy.the series toxin of rPEAwhich produce by gene-recombination technique can be expected soon as new-typeanti-cancer missile to be used for clinical. Finished product rPEA as medical new-type anti-cancer missile, its mechanism and assay method of ADP-RibosyltransferaseActivity have no reportage in our country.[Objective I The assay method ofADP-Ribosyltransferase Activity will beestablished. ADP-Ribosyltransferase Activity of three rPEA which construction,expression, purification and production by our laboratory will be have assaied andexhibited. Changement of ADP-Ribosyltransferase Activity is assaed for LRH-PE4Oduring purification process..imethodi 1. Eukaryotic Elongation factor-2s(EF-2) will be maked and chose byWheat Germ and the lasate from reticulocytes of Rabbit and sheep . 2.Theradioenzymatic assay method for ADP-Ribosyltransferase Activity of rPEA will beestablished that to use radio isotope[14C1 label NAD substrate 3. ADP-Ribosyltransferase Activity of three rPEAs will be detected.[Result]I .EF-2 of content ( >3Opmo]IlOul ) and radioactivity(589.Ocpm/lOul) were hightest which from the lasate of sheep reticulocytes inthat EF-2s were maked from three aniso-source (Wheat Germ and the lasate fromreticulocytes of Rabbit and sheep) 2. Radioactivity of ADP-RibosyltransferaseActivity which supernatent of thallus lasates to use engineering fungus ferment frome3three rPliA~ were constructed by our laboratory were EGF-PE4O 101.0 cpm4igtGLPE38 :j 13.8 cpmlpg LRH-PE4O :126.3 cpmljag 3. Supernatent of thalluslasate to use engineering fungus ferment frome LRH-PE4O was purified, removalratio of the excresccnt protein was 99.88%, the specific activity was heightented froml26.3cpm4~g to l2818.4cpm4~g in fore and after purification ,the multiple ofpurification was 101.5.[Conclusion] 1. EF-2 which was produced by the las ate from sheep reticulocytesreplacement Wheat Germ and the lasate from Rabbit reticulocytes is feasible 2. Theradioenzymatic assay method was establishcd for ADP-Ribosyltransferase Activity ofrPEA, there was good repeatability and high precision ?that is a assay methodpractical and reliable by this experiment for monitoring quality index of rPEApreparation agent 3.There were ADP-Ribosyltransferase Activity evident that threerPEA were constructed by our laboratory(EGF-PE-40. LRH-PE-40. EGF-PE-38) prompting ,the mechanism of series toxin of rPEA coincide with natural PEA 4.After purification , the specific activity of ADP-Ribosyltransferase Activity of LRH-PE4O reached (12836.0 cpm4tg) that outrange 6.3 multiple from refiner standard ofnatural PEA (1753 cpm]~.tg) (electrophoresis rank) 憈he rosy prospect was showedthat finished product rPEA was used for treating tumoure as medical new-type anti-cancer missile.