Isolation And Purification Of Disintegrin In The Venom Of Gloydius Blomhoffi Brevicaudus

Researchers exerted great fascination on the snake venom due to its diverse biological activities. Until now, several proteins with potential clinical values in snake venom have already been isolated. Among them, disintegrins are a family of small proteins mainly derived from snake venoms. Most of the disintegrins contain RGD or KGD sequences, which are the structural motifs recognized by the platelet fibrinogen receptorαIIbβ3, and also act as potential antagonists of several integrins includingαVβ3 andα5β1 which are expressed on vascular endothelial cells and some tumor cells. In addition to their potential antiplatelet activity, studies of disintegrins such as Rhodostomin[1], Accutin[2], Salmosin[3] [4], Saxatilin[5] and Contortrostatin[6] etc., have revealed a new application in inhibiting angiogenesis and tumor growth [1-9].Integrins are a family of heterodimeric receptors on cell surface. The known 18αand 8βsubunits combine to form at least 24αβheterodimers[10-12]. Most cells express more than one type of integrin heterodimers. Integrin expression profiles are unique for distinct cell types, and change with development stage and physiological conditions within a cell type. The common binding site of ligands is composed of the N-terminal ofαandβsubunits. Because most of cell integrins recognize RGD sequence and most of ECM molecules contain the sequence RGD, the RGD sequence has been already considered as the binding site of ECM molecules to integrins[13]. Although the intracellular part of integrins is much shorter, they can form links to the cytoskeleton throughα-actinin, talin and vinculin. Integrins are the major family of cell adhesion receptors that mediate cell adhesion to the extracellular matrix (ECM). Integrin-mediated adhesion and signaling play essential roles in cell adhesion, migration, and morphogenesis during cancer metastasis[14].rAdinbitor had been cloned from Gloydius blomhoffi brevicaudus and characterized as a novel snake disintegrin. In the study, we designed two oligonucleotide primers. Total RNA was extracted from venom gland and cDNA with 219 bp long was produced by RT-PCR. The encoded protein composed of 73 amino acids includes 12 cysteine residues and an RGD motif, the signature motif of disintegrin. Subsequently, rAdinbitor with molecular weight of 9kD was expressed in E.coli and purified with His-Bind affinity chromatography. Shown by related experiments, rAdinbitor has obvious and bracing effects on inhibiting ADP-induced platelet aggregation as well as migration and proliferation of tumor cells.In order to compare the structures and functions between rAdinbitor and the native disintegrin in the snake venom and perfect the techniques of producing the disintegrin, my job focuses on isolating and purifying natural disintegrin from the venom of Gloydius blomhoffi brevicaudus.(1) The disintegrin in venom of Gloydius blomhoffi brevicaudus was isolated and purified by gel filtration and reverse phase-high performance liquid chromatography (RP-HPLC). The activity of disintegrin was detected by the assay of inhibition on platelet aggregation. The result indicated that the coupling of the two kinds of chromatography was an effective approach for isolation and purification of disintegrin from the venom of Gloydius blomhoffi brevicaudus. The result of silver stain, after Tricine SDS-PAGE, showed two clear, detached protein bands with molecular weights of about 17kD and 12kD respectively.(2) The disintegrin in venom of Gloydius blomhoffi brevicaudus was tried to be isolated through gel filtration and ion exchange chromatography. The activity of disintegrin was detected by the experiment of inhibition on platelet aggregation. As a final result, there are seven protein bands shown by silver stain. Furthermore, two bands of protein both of which are not separated completely appear within the arrange of molecular mass where the disintegrin should emerge expectedly.To sum up, this research successful isolated and purified the disintegrin in venom of Gloydius blomhoffi brevicaudus through using the combined approach of two types of chromatography?gel filtration and RP-HPLC. This technique can serve to producting purified disintegrin for further research such as detecting structures and functions of the natural disintegrin in the venom of Gloydius blomhoffi brevicaudus and making the comparison with rAdinbitor.