The Prokaryotic Expression, Purification And Biological Effect Of The Antibody DPK3-scFv Target To DNA-PKcs

Malignant tumor is the killer which threaten human life and health,and radiotherapyis one of the most important means for malignant tumor therapy.However, radiotherapyinduced severe adverse effects resulted from the damage in normal tissues around thecancer,and radiation-toleration of tumer cells, which lead to the limitation for theclinical practice of radiotherapy.Therefore,it is an important task in the field of radiationbiology and oncology how to increase the sensitivity of cancer cells to radiotherapy.The lethal effect which resulted from radiotherapy to tumor cells,mostly oweing toDNA double strand breaks(DSBs) induced by ionizing radiation(IR),induces to celldeath and other genomic events after irradiation. Cells can repair the DSBs to ensure thegenomic stability.The cell will manifest radiation tolerance if the cell have thesupra-capability to repair the DSBs.Eukaryotic cells have two chief DSBs repairmechanistic pathways,including the homologous recombination(HR) andnon-homologous end joining(NHEJ).And DNA-dependent protein kinase(DNA-PK) isa crucial component in NHEJ.DNA-PK which belongs to Phosphatidylinositol-3-kinase-like kinase(PIKK)superfamily,is composed of the regulatory component,Ku70/Ku80subunits,and theDNA-dependent protein kinase catalytic subunit(DNA-PKcs).DNA-PKcs produces amarked effect in DSBs repair mainly through its autophosphorylation andphosphorylating other targets.DNA-PKcs can take part in cell cycle G2regulation,cellapoptosis,autophagic death,telomere length maintenance and V(D)J recombination.Inaddition,it was reported that DNA-PKcs is highly expressed in various cancer tissues,and activity of DNA-PKcs increases in tumor cells associated withhigh-metastatic or radio-resistance.sensitivity of tumor cells to IR will increase,ifDNA-PKcs is deficient;so DNA-PKcs has the significant value as a target of increasingsensitivity of tumor cells to IR. At present there are diverse inhibitors to inhibit theactivity of DNA-PKcs,but these inhibitors either show low specificity or induction ofhepatotoxicity or induction of immunological reactions,which restrict their practicalapplication.now,the development of human-derived antibody technique,especially thephage antibody library technique,the screening of single-chain variable antibodyfragment(scFv) provides a new approach in tumor biological therapy.Single-chain variable antibody fragment(scFv),is a recombination protein composedof variable region of heavy chain(VH) and variable region of light chain(VL) through alinker including ten to fifteen amino acids;and it is the minimum antibody fragment tohave combining site of complete antigens.ScFv has a great of advantages:smallmolecule,low immunogenicity;easily getting in microenviroment around tumors;shorthalf-life,not easily accumulating;not Fc fragment,not easily binding non-target cells tohave Fc acceptors;easily proceeding genetic manipulation and genetic engineering.ScFv will develop enormous latent capacity in clinical diagnosis and therapy,and atpresent some anti-tumor svFv alreadly get into in vivo tests.Our laboratory successfullysieves specific human-derived single-chain antibody DPK3-scFv targeting to DPK3fragment(AA1960-2227) of DNA-PKcs from the human-derived phage antibodylibrary;The antibody genes were transfected into Hela cells,we find DPK3-scFvimproves sensitivity of tumors to IR.Our research assesses the transmembrane delivery and targeting stability ofDPK3-scFv,the effect of autophosphrylation and cell cycle because of DPK3-scFvthrough expressing the protein DPK3-scFv.Our research provides the evidence forsingle-chain antibody to have radiosensitization.The main results are as follows:1The target gene HA-DPK3-scFv was amplified,cloned into the prokaryotic expression vector pET28a,transformed into E.coli BL21(DE3).By optimizing theexpression conditions the single-chain antibody HA-DPK3-scFv was successfullyexpressed as inclusion bodies,and then we could denature、renature and purify theHA-DPK3-scFv.2Detect the protein activity of HA-DPK3-scFv by DNA-dependent protein kinaseanalysis system, HA-DPK3-scFv was expressed in a large-capacity and purified.Observe the transmembrane transport of HA-DPK3-scFv,results show that the singlechain antibody HA-DPK3-scFv did not cross the cell membrane into cells.3The target gene DPK3-scFv was amplified,cloned into the prokaryotic expressionvector pET28a,transformed into E.coli BL21(DE3).By optimizing the expressionconditions the single-chain antibody DPK3-scFv was successfully expressed in the formof soluble expression,purify DPK3-scFv by affinity chromatography in vitro and detectthe protein activity of DPK3-scFv by DNA-dependent protein kinase analysis system.4The single-chain antibody DPK3-scFv after expression and purification,wasadded to the serum-free medium at a final concentration of70μg/ml and incubated withtumor cells;Then assess the cell-penetrating delivery of DPK3-scFv at different timepoints by immunofluorescence staining and western blotting;The result indicated thatDPK3-scFv was efficiently introduced into cells cultivated with serum-free medium andmainly localized in the nucleus;its half life is about12h.5On account of the result that DPK3-scFv was introduced into cells after1h byincubation,cells incubated with DPK3-scFv2hours were treated4Gy γray;then wedetected the affect of DNA-PKcs/pS2056phosphorylation level at different time pointsby immunofluorescence staining and western blotting; The result indicated thatcompared with the control group(tumor cells incubated without DPK3-scFv),DNA-PKcs/pS2056phosphorylation level of tumor cells incubated with DPK3-scFvafter IR was down-regulated.In conclusion:we successfully expressed and purified the high purity protein DPK3-scFv,and detect its activity.DPK3-scFv could be effectively delivered into cellsand stably exist. Autophosphorylation of DNA-PKcs at ser-2056was found to bedown-regulated in tumor cells to IR by DPK3-scFv.