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Expression of Kininogen D560-148 TRAIL114-281 fusion protein and its angiogenesisinhibiting and apoptosisinducing effect
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Abstract:
Objective:To express Kininogen D560148TRAIL114281 fusion protein using prokaryotic system and observe its biological functions.Methods: The Kininogen D560148 gene and TNFrelated apoptosisinducing ligand (TRAIL114281 ) gene were amplified by PCR and were cloned into pMAL expression vector to construct recombinant pMALKininogen D560148 (pMALKD5), pMALTRAIL114281 (pMALTRAIL) and pMALKininogen D560148TRAIL114281 (pMALKT) plasmids, respectively. The plasmids were transformed into E. coli BL21 and were efficiently expressed after IPTG induction. The purified MBPKD5, MBPTRAIL and MBPKT proteins were obtained by amylose resin affinity purification column. The proliferation of cells was measured by MTT; tube formation of endothelial cell was detected by tube formation assay; and the apoptosis of cells were observed by electron microscopic and FCM.Results: Prokaryotic expression vectors pMALKD5, pMALTRAIL and pMALKT and their purified fusion proteins MBPKD5, MBPTRAIL and MBPKT were successfully obtained. MBPKT significantly inhibited the proliferation of ECV304 endothelial cells, SW1990 pancreatic cancer cells and the tube formation of ECV304 cells compared with those of MBPKD5 and MBPTRAIL. Meanwhile, MBPKT dosedependently induced the apoptosis of SW1990 cells.Conclusion: Kininogen D560148TRAIL114281 fusion protein can inhibit the proliferation of tumor cells and angiogenesis of endothelial cells, which lays a foundation for further research on tumortargeting drugs.
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Supported by the Modern Industrial Development Foundation for Biology and New Drugs of Shanghai (No. 024319115)
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