Preparation And Identification Of CPPS-β-glucosidase Conjugates And Activation In Vitro Amygdalin Preliminary Research To Kill Bladder Cancer Cells

Objective:Discusses application of N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP)to prepare CPPs-β-glucosidase conjugates feasibilty and identification of itactive, conjugating way. And preliminary studies in CPPs-β-glucosidaseconjugates with coupling amygdalin vitro killing effect on human bladder cancerEJ cells. In order to further build efficiency in CPPs-β-glucosidase-antibodyenzyme prodrug system to provide the experimental basis.Methods：1、Revive after our early experiment building and identification of sequencingthe high expression of CCPs-EGFP strains of e. coli and a large number ofculture, induced expression of fusion protein. Using the reduced polyacrylamide(SDS-PAGE) was carried out on the size of fusion protein molecular weight andpurity identification. Using the method of BCA protein concentrationdetermination kit measuring the concentration of purified fusion protein. Thefusion protein with bladder cancer EJ cell incubation together, on fluorescencemicroscope, identify its acrossing activity2、CPPs-EGFP andβ-glucosidase were crosslinked by SPDP and purified by Ni-NAT Superflow Cartridge first and next by Sephadex G-75columnchromatography.CPPs-β-glucosidase conjugates were tested by reducing andnon-reducing SDS－PAGE assay. CPPs-β-glucosidase conjugates and EJcells were incubated together to detect the penetrating activity with fluorescencemicroscope.The enzymatic activity of the CPPs-β-glucosidase conjugates weretested by the beta-glycosidase enzymes text box(DBGD-100).3、MTT method is used to determine CPPs-β-Glucosidase Conjugates andactivation in vitro amygdalin preliminary research to kill bladder cancer cells.Result:1、A lot of CPPs-EGFP was expressed and purifyied and the CPPs-EGFP issoluble, green fusion protein. Identified by reduced SDS-PAGE the CPPs-EGFPis about31kd molecular weight, high purity, no obvious impurity band.Identified with the method of BCA protein concentration determination kitmeasuring the concentration of purified fusion protein and the Concentration is10.72㎎/ml. The fusion protein with bladder cancer EJ cell incubationtogether after60minutes, bladder cancer EJ cells was observed underfluorescence microscope with green fluorescence.2、CPPs-β-glucosidase conjugates by non-reducing SDS－PAGE assayshowed protein band at high molecular weight end,but showed protein bandscorresponding to the sites of CPPs-EGFP andβ-glucosidase by reducing SDS－PAGE assay.The EJ cells which were incubated with CPPs-β-glucosidaseconjugates showed green fluorescent.The CPPs-β-glucosidase conjugates keptenzymatic activity tesed by colorimetry assay. The enzymatic activity is about80%of the same concentration of beta-glycosidase enzymes.3、The determined by MTT method, CPPs-β-Glucosidase Conjugates activatein vitro amygdalin to kill bladder cancer cells,the inhibitory effect is Stronger then pure beta glycosidase enzymes.Conclusion:Expressed and purified a lot of CCPs-EGFP Successfully. CPPs-β-glucosidase conjugates can be prepared by the method of SPDP conjugationtechnology successfully. Respectively by Ni-NAT Superflow Cartidge andSephadex G-75chromatographic column CPPs-β-glucosidase conjugates canbe successfully purified.And the CPPs-β-glucosidase conjugates still keepgood through activity and enzyme activity after purified. CPPs-β-glucosidaseconjugates activate amygdalin in vitro can increase the role of kill bladdercancer EJ cells.This method may be used to sloue the problem that enzymeprodrug system molecular weight is bigger and enter the cell difficulty. Thismethod may be for further build efficiency in CPPs-β-glucosidase-antibodyenzyme prodrug system to provide the experimental basis.