Prokaryotic Expression Of Recombinant Disintegrin From Gloydius Brevicaudus Venom And Its Biological Activity Identification

Objective:To construct pET21a（+）expression vector containing gene of disintegrin/DI,which was transformed into E.coli origami B（DE3）competent cells then.Purification was accomplished by nickel affinity chromatography column and r-DI could lay a foundation for later bioactivity determination,structural relationship research and anti-tumor activity application in vivo.Methods:1.Construction of the expression vector（1）Synthesis of cDNA:Based on amino acid sequence of native disintegrin,the cDNA of disintegrin was optimized and synthesized and the nucleotides encoding Nde I and Xho I were added.（2）Construction of vector:The cDNA was ligated into pET21a（+）vector and transformed into E.coli T1.2.Expression and purification of the recombinant disintegrin（1）Transfer of vectors:Recombinant plasmids containing gene DI were transferred into E.coli origami B.Correct clone was confirmed by a Nde I/Xho I set of restriction enzymes and nucleotide sequencing.（2）Exploration of expression conditions:To explore the effect of 2YT or self-induced medium on expression of recombinant disinterrin and determine the optimal expression conditions.（3）Purification of recombinant disinterrin:The proteins were cleaved and eluted from affinity nickel column.3.Determination of the purity and immunology activity of recombinant disintegrins Recombinant disintegrins were analyzed by Tricine-PAGE gel electrophoresis under reducing conditions to determine its molecular weight and purity.Western blot analysis using His-tag antibody and anti-Gloydius brevicaudus venom serum respectively were used to verify the correct expression of Recombinant disintegrins.4.Determination of the biological activity of recombinant disintegrins（1）Antiplatelet aggregation activity assay:The inhibition of ADP-induced platelet aggregation was confirmed by r-DI in PRP.（2）Inhibition of tumor cell growth,adhesion,migration,invasion and inhibition of tubulogenesis activity assay:The viability of B16F10 cells and cell adhesion were done by MMT assay.Angiogenesis ability was determined by tube formation assay.Cell migration was detected by wound healing assay.Cell invasion was tested by Transwell assay.（3）Surface plasmon resonance（SPR）:To detect the interaction with integrinαⅡbβ3 and determine the kinetic constant（Ka,Kd）and the affinity constant（KD）.Results:1.The cDNA of recombinant disintegrins was a 219 bp long fragment coding for 69 amino acids and the Nde I and Xho I sites were added at both ends,pET21a（+）-DI expression vector was costructed successfully,which could express protein with C-end containing His sequences.2.Determined by restriction enzymes and nucleotide sequencing,plasmids containing gene DI were transferred into E.coli T1 and origami B successfully.3.In ZYD medium,induction temperature of 23℃and induction time of20h is the optimal condition for the expression of recombinant disintegrin（r-DI）.4.After r-DI was purified from crude lysate by affinity nickel column,a yield of 10 mg of r-DI was obtained.r-DI had a protein band at about 8.7 k Da and it’s purity reached 95%above after analyzed by Tricine-PAGE gel electrophoresis.Western blot analysis using His-tag antibody and anti-Gloydius brevicaudus venom serum respectively indicated that the recombinant disintegrin is reactive with both antibodies.5.r-DI exerted an dose-dependent inhibitory effect on ADP-induced platelet aggregation.At concentrations of 1.4,2.9,and 5.9μM,the inhibition rates of r-DI were 28%,53%,and 91%respectively.6.r-D exhibited an dose-dependent inhibitory effect on proliferation of B16F10 cells with an IC₅₀of 1.59μM,and reduced the adhesion on matrigel,the number of small tubes,migration ability and invasion effect with a dose-dependent manner.At concentrations of 0.12,0.58,1.15μM,r-DI restrained 20.3%,39.7%,50.2%of cell adhesion,and suppressed 31.7%,49.3%,71.1%of cell invasion.7.r-DI can specifically bind to integrinαⅡbβ3,the binding constant of r-DI and integrinαⅡbβ3 is 4.23x10^-3mol/L,dissociation constant is 5.91x10^-3mol/L and the affinity is 1.40x10^-6mol/L.Conclusion:1.Disintegrins found in venomous snakes can be expressed in E.coli cells and further purified by one step chromatography with high yield and purity.2.Recombinant disintegrin inhibited ADP-induced platelet aggregation,tumor cell adhesion,angiogenesis,migration,and invasion.3.Recombinant disintegrins can specifically combine with the integrinαⅡbβ3,competitively blocking the receptors.