Cloning, expression and bioactivity analysis of human granulysin
- VernacularTitle:颗粒溶素的克隆表达及活性分析
- Author:
Wanxia WANG
;
Xi LAN
;
Xianghong XU
;
Jun JU
;
Jixing LIU
- Publication Type:Journal Article
- Keywords:
granulysin;
prokaryotic expression
- From:
Basic & Clinical Medicine
2010;30(1):75-79
- CountryChina
- Language:Chinese
-
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-GN-LY9K and pET-GNLY15K were transferred to E. Coli Rosetta (DE3). The fusion protein was identified by SDS-PAGE and Western blot. The bioactivity of granulysin fusion protein was measured by MTT assay. Results The prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K were successfully constructed. The corresponding protein was highly expressed in E. Coli. Recombinant protein was specifically bound by anti-granulysin antibody. GNLY9K fusion protein significantly inhibited the growth of A549 cells in a dose-dependent manner, while GN-LY15K had little effect on the growth of A549. Conclusion Granulysins with different mw were successfully expressed using prokaryotic expression system, which might be helpful for the further study of granulysin.
