The Expression Of A Recombinant Disintegrin Of Gloydius Brevicaudus Venom And Determination Of Its Biological Activities

Objective Disintegrin of Gloydius brevicaudus venom and human IgG1 Fc gene were cloned to obtain a recombinant fusion gene DI-Fc.The recombinant eukaryotic expression plasmids pXC17.4/pCDAN3.1(+)-rDI were constructed and transfected into 293 cells to express recombinant disintegrin.The expression of the recombinant disintegrin lay a foundation for researching its activities,structure activity relationship and clinical application.Methods 1 Construction of the recombinant expression vector 1)Template synthesisDI: The cDNA sequence of disintegrin was sequenced and synthesized according to the native disintegrin which was purificated from Gloydius brevicaudus venom.In addition,there is a IgG3 Upper Hinge at the disintegrin 3' domain.IgG1 Fc: According to NCBI IgG1 Fc gene sequence,We synthesized IgG1 CH2CH3 area.2)Primer designDisign primers according to the above sequences and another pairs of primers to replace the IgG3 Upper Hinge with IgG1 Hinge.3)MDF/DDF fusion geneThe above two pieces were gained by PCR amplification.Through Overlap PCR,disintegrin gene was fused with IgG1 Fc gene and then we get the DI-Fc fusion gene.Finaly we obtained two styles fusion gene,and named them MDF/DDF which hinge domain was IgG3 Upper Hinge and IgG1 Hinge respectively.MDF/DDF fusion gene were subcloned into T Vector pMD19 through TA clone.HindIII/EcoRI restriction enzyme digestion and sequencing were used to identify the correct clone.4)Construction of the recombinant expression vectorMDF/DDF fusion gene were cloned into eukaryotic expression vector pXC17.4/pCDAN3.1(+).Then transfer them into competent E.coli DH5? cells.Hind III/EcoR I restriction enzymes digested the four clones.2 Expression and purification of the recombinant disintegrin 1)The four expression vectors were pretransfected into 293 FT cells with Lipofectamine 2000.2)The quantity of MDF/DDF proteins which were expressed in cell supernatant was determined through ELISA so that we could select the optimal expression vector.3)The recombinant disintegrins MDF and DDF were obtained by two methods: they were transfecting the 293 FT cells with PEI respectively or transfecting the Expi293 F with ExpiFectamine293 reagent respectively.The quantity of MDF/DDF with the two methods was compared.4)HiTrap MabSelect SuRe ProteinA affinity chromatography purified the cell supernatant.3 Analysis purity of the recombinant disintegrinsUPLC was used for analyzing the purity and molecular weight of the recombinant disintegrins.Chromatographic condition: SEC liquid chromatographic column(4.6mm×150mm,1.7?m,Waters);temperature column:30?;mobile phase: 0.02 M PB+0.15 M NaCl buffer;detector: TUV;sample size: 10?l;flow rate: 0.4mL·min-1;UV detection wavelength: 280 nm.4 Determination the immunology and biological activity of recombinant disintegrins 1)Recombinant disintegrins were analiyzed under both reducing and non-reducing conditions SDS-PAGE gel electrophoresis and Western blot which was incubated HRP anti-Human IgG(Fc specific)antibody and anti-Gloydius brevicaudus venom serum repectively to verify their expression.2)Platelet aggregation test(PAgT)detected its biological activity.Results 1 Construction of the recombinant expression vectors 1)Fusion gene MDF/DDF The PCR products of Disintegrin gene was 358bp(349bp),the PCR products of IgG1 Fc was 661bp(644bp),the fusion gene MDF was 970 bp and DDF was 985 bp.MDF/DDF fusion gene were subcloned into T Vector pMD19 through TA clone.The correct clones were gained by HindIII/EcoRI restriction enzyme digestion and sequencing.2)Recombinant expression vectors Restriction analysis proved that recombinant plasmid pXC17.4-MDF/DDF,pCDAN3.1(+)-MDF/DDF was constructed correctly.2 Expression,purification and purity assay of recombinant disintegrins 1)Expression of recombinant disintegrins MDF/DDF proteins which were expressed in 293 cell supernatant were positive by the four vectors,pXC17.4 as the expression vector: MDF,DDF expression quantity was 0.68mg/L,0.97mg/L repectively;pCDNA3.1(+)as the expression vector: MDF,DDF expression quantity was 4.3 mg/L,2.3mg/L repectively.So the pCDAN3.1(+)-MDF/DDF expression vectors were used to expression recombinant disintegrins in batch.2)Purification of recombinant disintegrins A single peak of MDF/DDF protein was obtained through HiTrap MabSelect SuRe Protein A chromatography.Expression quantitation of the proteins is about 2mg/L with PEI and 45mg/L with ExpiFectamine reagent.3)Purity assay of recombinant disintegrins UPLC showed that the molecuar weight of MDF and DDF were greater than 180 kDa exist in a multimer form.There was some low molecuar weight proteins in MDF.3 Determination the immunology and biological activity of recombinant disintegrins 1)Immunoassay SDS-PAGE gel electrophoresis showed that there were molecuar weight about 35 kDa and 70 kDa band in MDF and DDF lane respectively,but there were more multimer in the two designs.And in Western analysis probed by HRP anti-Human IgG(Fc specific)antibody and anti-Gloydius brevicaudus venom antibody repectively indicated that the recombinant disintegrins is reactive with both two antibody while there is no reactivity with the isotype IgG.So the SDS-PAGE gel electrophoresis and Western consistently suggesting that the recombinant proteins were a fusion of disintegrin and Fc.2)Platelet aggregation assay Platelet aggregation test revealed that the recombinant disintegrin has the activity of inhibiting platelet aggregation induced by ADP with a dose-dependent manner.ConclusionThe recombinant expression vectors pXC17.4-MDF/DDF,pCDAN3.1(+)-MDF/DDF was successfully constructed by DNA recombination technology and were expressed in 293 cells.And the recombinant disintegrins had the activity of inhibiting platelet aggregation induced by ADP with a dose-dependent manner.The expression of disintegrin and identification of its functional activity lay the foundation for further study on its role in anti-thrombus,inhibiting tumor cell adhesion,migration and tumor angiogenesis.