颗粒溶素的克隆表达及活性分析

Cloning,expression and bioactivity analysis of human granulysin

目的克隆人颗粒溶素基因并进行原核表达。方法体外分离并培养外周血单个核细胞,抽提总RNA,RT-PCR扩增人颗粒溶素的基因片段,并将其插入pMD18-T进行测序,鉴定正确后分别将目的基因亚克隆至pET32a(+),构建原核表达质粒pET-GNLY9K和pET-GNLY15K,将重组质粒转入大肠杆菌Rosetta(DE3),IPTG诱导表达融合蛋白,SDS-PAGE和Western-blot鉴定融合蛋白的正确性。MTT检测颗粒溶素融合蛋白的生物活性。结果成功构建了原核表达载体pET-GNLY9K和pET-GNLY15K,经诱导在原核表达系统中高效表达了相应分子质量约31和37ku的融合蛋白,重组颗粒溶素融合蛋白能特异地与抗颗粒溶素抗体结合,GNLY9K融合蛋白可以明显抑制A549细胞增殖,而GNLY15K融合蛋白对细胞增殖影响极低。结论利用原核表达体系成功地表达了不同分子质量的颗粒溶素融合蛋白,为颗粒溶素的后续研究奠定了基础。

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. eoli 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 pro- karyotic 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 Ag49 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.