Clone And Functional Analysis Of A New Snake Venom Metalloproteinase/disintegrin Gene

Snake venom metalloproteinases/disintegrins, which contribute to the hemorrhage activity of snake venom, was one of the major functional peptides of snake venom. More than 20 sequences of SVMPs(snake venom metalloproteinases) have been detected by now, and 3D structure of four SVMPs have been illustrated. Disintegrins was a kind of small cysteine-rich peptides which can inhibit platelet aggregation, the cDNA sequence data showed that the disintegrins and the PⅡ SVMPs belong to one pro-protein.We cloned a gene fragment which contain a disintegrin domain from Agkistrodon halys by RT-PCR methods, use the distinguish sequence of this fragment as primer we clone the full length gene. The sequencing data revealed that this gene is a new member of P-Ⅱ class SVMP, its deduced amino acid sequence has a high similarity with other SVMPs(contortrostatin 78%,bothrostatin 77%), it has two additional cysteine (Cys407 and Cys426) in the disintegrin domain than other RGD contain SVMPs, we named this gene Agksitin to distinguish from other SVMPs.Agkistin-s (amino acid 344-488,the small gene fragment that consists of space region and the complete disintegrin domain) was insert to the pET-28a(+) vector, soluble Agksitin-s protein was expressed after the E.coli was induced by IPTG at 30℃, then the expressed protein waspurified through Ni2+-IDA column. At the same time, the Agkistin and Agksitin-s gene were inserted to the pFastBac-Htb和pFastBac-Hta vector of the recombinant baculovirus system (Bac-to-Bac system),respectively, then they were expressed in Tn-5B1-4 cell. After purified by Ni2+-IDA column, we obtained pure Agkistin and Agksitin-s protein. These results revealed that the disintegrin domain contribute to the angiogenesis inhibit activity of Agkistin.The platelet aggregation inhibit activities of Agkistin and Agkistin-s protein (expressed in Tn cell) were measured, data showed the Agksitin-s protein have some disaggregate activity under high concentration. At the same time, we analyzed the effect of Agkistin and Agksitin-s on the angiogenesis by the chicken CAM assay, and find that they can inhibit the spontaneous angiogenesis of chicken CAM.The apoptosis of endothelial cell plays a critical role in many physiological events such as the angiogenesis. We find the Agkistin protein can induce the micro-vessel endothelial cell HMEC apoptosis by adding the protein into the cultured HMEC cells, this activity was measured by flow cytometry,data showed 0.5μM Agksitin can induce 34.07% HMEC cell apoptosis. Furthermore, the Agksitin-s can also inhibit the BAEC cell growth.