基础医学与临床 ›› 2010, Vol. 30 ›› Issue (1): 75-79.

• 研究论文 • 上一篇    下一篇

颗粒溶素的克隆表达及活性分析

王晚霞 兰喜 徐向红 居军 柳纪省   

  1. 甘肃省人民医院 中国农业科学院兰州兽医研究所 甘肃省人民医院 甘肃省人民医院 中国农业科学院兰州兽医研究所
  • 收稿日期:2009-02-13 修回日期:2009-05-06 出版日期:2010-01-05 发布日期:2010-01-05
  • 通讯作者: 居军

Cloning, expression and bioactivity analysis of Human Granulysin

Wan-xia WANG, Xi LAN, Xiang-hong XU, Jun JU, Ji-xing LIU   

  1. Gansu Provincial People's Hospital Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences Gansu Provincial People's Hospital
  • Received:2009-02-13 Revised:2009-05-06 Online:2010-01-05 Published:2010-01-05
  • Contact: Jun JU,

摘要: 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37kD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。

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