Production And Anti-glioma Effects Of Recombinant Immunotoxin EphrinA1-PE38KDEL

Background and ObjectvieGlioma is the most frequent tumor in the central nervous system.The mortality of glioma remains high despite multi-modal tumour therapy including surgery,radio-and chemotherapy.Novel therapeutic strategy for glioma is urgently needed.Recombinant Immunotoxin is a popular method in molecular targeting therapy and a promising method for treatment of glioma.Recombinant immunotoxins consist of two major components:tumor-specific targeting molecules and toxin proteins.EphA2 was reported to be highly expressed in glioma tissue,however expressed at a low level in normal tissue.we select EphrinA1,the specific ligand of EphA2,as the target molecule and PE38KDE as toxin protein.We establish the recombinant immunotoxin EphrinA1-PE38KDEL via a prokaryotic system and evaluate its therapeutic effect on aggressive glioma.MethodsWe synthesize a sequence encoding a recombinant immunotoxin EphrinA1-PE38KDEL by DNA cloning and mutagenesis.We construct the recombinant immunotoxin vector plasmid by designing the target gene primer design,plasmid digestion,ligation of the target gene and plasmid,and transformation of the vector plasmid with the cloned strain.We prokaryotically express the recombinant immunotoxin EphrinA1-PE38KDEL by using IPTG induction,Ni-NTA affinity chromatography,imidazole gradient and pH gradient elution purification experiments.We measure the concentration of EphrinAl-PE38KDEL by BCA protein assay.We perform an intake assay to evaluate the specific recognition of EphA2 by EphrinAl-PE38KDEL and entry into the glioma cells.We use CCK-8 assay to evaluate the in vitro effect of EphrinAl-PE38KDEL on glioma cells and HEB.ResultsWe successfully construct the sequence encoding EphrinAl-PE38KDEL and confirm it by double digestion with PCR and gene sequencing of vector plasmids.We confirmed by SDS-PAGE electrophoresis and Western blotting experiments that E.coli BL21(DE3)transformed with recombinant immunotoxin vector pET2 8 a-EphrinA1-PE3 8KDEL can express recombinant immunotoxin EphrinAl-PE38KDEL.The concentration of the soluble recombinant immunotoxin EphrinAl-PE38KDEL,expressed and purified by prokaryotic expression,was 0.15?g/?l as measured by the protein BC A method.We observed that recombinant immunotoxin EphrinAl-PE38KDEL can enter glioma cells via ligand receptor binding reactions by using cellular uptake assay.We found that the recombinant immunotoxin EphrinAl-PE38KDEL has a targeted killing effect on human glioma cells U251 and U87,and the effect is dose dependent,however,we found no significant killing effect on human normal brain astrocytes HEB.ConclusionThe recombinant immunotoxin EphrinAl-PE38KDEL produced by prokaryotic expression has an inhibitive effect on glioma cells without affecting the growth of normal brain glial cells.