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• 生物工程 •    下一篇

地皮菜中一种新型水应激蛋白基因的克隆表达及其对结肠癌细胞的抑制作用

郭松佳,单树花,晋小婷,李宗伟,宋莉,李卓玉   

  1. 山西大学生物技术研究所
  • 收稿日期:2013-09-25 修回日期:2014-06-23 出版日期:2014-07-15 发布日期:2014-07-18
  • 通讯作者: 李卓玉 E-mail:lzy@sxu.edu.cn
  • 基金资助:
    牛乳骨桥蛋白与乳铁蛋白协同成骨作用及热处理对其影响研究;高等学校博士学科点专项科研基金;山西省2011海外人才基金

Cloning,expression of a novel water stress protein from Nostoc commune Vauch. and its growth inhibitory effects on colon cancer cells

  • Received:2013-09-25 Revised:2014-06-23 Online:2014-07-15 Published:2014-07-18

摘要: 摘 要:利用PCR扩增出地皮菜中一种新型水应激蛋白(water stress protein ,WSP)的基因(Gene bank 序列号:KF003026),连接到原核表达载体pET28a上,测序鉴定后转化入大肠杆菌BL21,获得一种新型水应激蛋白表达工程菌;经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达及镍离子亲和层析柱(Ni-NTA)纯化后,得到了带6×His标签、分子量大小为40kD的可溶性目的蛋白,称其为water stress protein 1(WSP1)。MTT实验结果表明,WSP1蛋白可以抑制结肠癌细胞SW480的生长,而对正常结肠上皮FHC细胞无明显作用;DAPI细胞核染色结果表明,该蛋白能够诱导SW480细胞凋亡小体的形成;Western blot结果显示,WSP1蛋白能够促进procasepase-3和procaspase-8的活化剪切。以上结果表明,WSP1蛋白能够明显的抑制结肠癌SW480细胞的生长,其机制与其诱导该细胞凋亡有关。

关键词: 【关键词】 地皮菜, 水应激蛋白, 重组蛋白, 表达与纯化, 结肠癌, 凋亡

Abstract: Cloning,expression of a novel water stress protein from Nostoc commune Vauch. and its growth inhibitory effects on colon cancer cells Abstract: Water stress protein (WSP) gene was amplified by PCR from the Nostoc commune Vauch. genome (Gen bank NO. KF003026), and cloned into the prokaryotic expression vector pET28a. After being sequenced, the recombinant plasmid pET28a-WSP was transformed into E.coli BL21, and protein water stress protein 1(WSP1) was induced to express by isopropyl-β-D-thiogalactoside(IPTG). The soluble 6×histidine (His)-tagged fusion WSP1 was further purified by Ni+-NTA. The molecular weight of fused WPS1 was 40kD as expected. When applied to cells in culture, WSP1 could significantly suppress the growth of SW480 colon cancer cells, but had a negligible effect on that of FHC normal colon epithelial cells. In addition, WSP1-treated SW480 cells displayed the apoptotic characteristics of chromatin condensation and typical fragmented nuclei, as demonstrated by DAPI staining. WSP1 could also activate procaspase-8 and procaspase-3, resulting in their cleavage into active fragments. Taken together, WSP1 has shown obvious anti-colon cancer SW480 cells effects, and the mechanism is associated with induction of apoptosis.

Key words: 【Key words】Nostoc commune Vauch., water stress protein, recombinant protein, expression and purification, colon cancer, apoptosis

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