The Comparison Activities Among The Lj-RGD3Toxin Protein And RLj-116

rLj-RGD3, is RGD toxin protein with3RGD motifs and17His (histidine) from thesaliva gland of Lampetra japonica.According to the literature, dinintegrin containing KGD(Lys-Gly-Asp) motif (disintegrin) can be potent and specifically binds to the platelet surfaceglycoprotein GPIIb/IIIa, αVβ3but highly vascular endothelial cell surface bindingexpression weak, This feature makes the KGD motif-containing protein toxin having aspecific effect against the platelet aggregation，but the effect against angiogenesis is lower. Inorder to further study of the relationship between the function and the structure of this RGDtoxin protein, we partially or totally changed the RGD motifs into KGD motifs using differentways of mutation. we used deleted mutation to change the RGD motifs from the wild typerLj-RGD3to be KGD motifs. And KGD is mutants of rLj-RGD3rLj-116compared withwild-type protein antithrombotic and comparative study of antiangiogenic function Methods:With3KGD is replace3RGD genes The mutant gene was constructed in the plasmid vectorof pET23b, And by genetic engineering methods to obtain a purified protein rLj-116mutants,Mutant and wild-type proteins were compared anti-angiogenic and anti-thrombotic activity.Which angiogenesis experiments using human umbilical vein endothelial cells HUVEC as anin vitro model, six-day-old chick embryo chorioallantoic membrane as an in vivo model CAM,Antithrombotic experiments are using SD rats as a model, determination of the two proteinsin rat arterial thrombosis, venous thrombosis and platelet aggregation Also, for thepreparation of a wild-type rLj-RGD3monoclonal antibody. About rLj-116antibodypreparation of the follow-up work will be carried out in the laboratory. The results showedthat: MTT assay showed rLj-RGD3IC50of0.889μmol/L, while rLj-116IC50of5.29μmol/L, That rLj-116inhibition of endothelial cell proliferation is much lower than rLj-RGD3.migration and invasion experiments described: ability to inhibit the migration and invasion ofcells rLj-116is also weaker than rLj-RGD3.Angiogenesis experiments show: rLj-RGD3andrLj-116has the effect of inhibiting angiogenesis, but the inhibition of mutant rLj-116issignificantly lower than rLj-RGD3.the anti-thrombotic experimental results showthat:Wild-type rLj-RGD3and thrombosis rLj-116mutant proteins are more significant. ButrLj-116antithrombotic effect of the effective dose is one fifth of the wild-type rLj-RGD3,showed rLj-116is better. In addition, the successful rLj-RGD3monoclonal antibody.Conclusion: Three RGD (Arg-Gly-Asp) replace all KGD (Lys-Gly-Asp) of protein rLj-116antithrombotic more powerful, while inhibiting angiogenesis lower, Preliminary evidence that it is a specific anti-thrombotic and almost no side effects of anti-angiogenic drug candidate,This will provide a theoretical basis for its further drug development.