关键词: 颗粒溶素, 原核表达

Abstract: Objective To obtain recombinant human granulysin using prokaryotic expression system. Methods Total RNA was extracted from cultured PBMC. Granulysin gene segments were obtained with granulysin-specific primers by RT-PCR and then inserted into pET32a(+) plasmid. After identification by DNA sequence, pET-GNLY9K and pET-GNLY15K were transferred to E. coli Rosetta (DE3).Fusion proteins were expressed under induction of IPTG.The fusion proteins was identificated by SDS-PAGE and Western-blot.The bioactivity of granulysin fusion protein was measured by MTT assay. Results Restriction enzyme digestion and sequence analysis showed that prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed.The corresponding molecular weight of 31 and 37kD fusion protein were highly expressed in E. coli after induction. Recombinant proteins could specifically bind to anti-granulysin antibody.MTT assay analysis showed that GNLY9K fusion protein could significantly inhibit the growth of A549 cells in a dose-dependent manner,while GNLY15K had little effect on the growth of A549.Conclusion Different molecular weight granulysin were successfully expressed using prokaryotic expression system,which would be helpful for the further study of granulysin.

Key words: Granulysin, Prokaryotic expression