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Cloning, Expression of Human Restin and It′s Antiangiogensis Activity
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Abstract:
Objective: To clone human angiogenesis inhibitor restin (hRS), express fusion protein in E.Coli and determine its biological activity. Methods: Restin gene was amplified by RT-PCR from Chinese human placenta tissue, then inserted into plasmid vector pGEM-T and sequenced. Prokaryotic expression vector pGEX-hRS was constructed and fusion protein GST-hRS was expressed. After the fusion protein purified by affinity chromatography and digested by thrombin, the anti-angiogenic activity of restin was tested by chicken chorio-allantoic assay. Results: RT-PCR product is 564 bp, the result of DNA sequencing identified the PCR product with the cDNA encoded human restin (Genbank COL15A1), but the synonymous mutation in bases encoding Ser21 (TCT→TCG) and mutation in Ser→Thr82(ACA→TCA) were also discovered. The expressed protein size was 20 kD after isolated fusion protein digested by thrombin, it appeared the expressed restin had the power to inhibit angiogenesis. Conclusion: The successful cloning and expression of human restin lay the foundation for the therapy of solid tumors.
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