摘要
目的合成竹叶青素trigramin基因,在原核系统中表达、纯化,对其功能进行初步研究。方法缓慢退火-PCR法合成竹叶青素基因并克隆入原核表达载体;IPTG诱导表达;亲和层析法纯化重组蛋白;常规柱层析和HPLC纯化天然竹叶青素;Born法测定竹叶青素对血小板聚集的抑制能力;CCK-8法测定竹叶青素对细胞增殖的影响;划痕实验分析竹叶青素对细胞运动的影响。结果合成了竹叶青素基因并在大肠杆菌中表达;获得纯度为89.1%的重组竹叶青素;重组竹叶青素抑制血小板聚集的IC50为0.78μmol.L-1,而天然竹叶青素为0.35μmol.L-1;竹叶青素不能抑制肿瘤细胞增殖,但可抑制细胞体外运动能力。结论本研究成功合成竹叶青素基因并在大肠杆菌系统进行了有功能的表达和纯化,竹叶青素可抑制肿瘤细胞体外运动。
Aim To synthesize trigramin gene, to per- form E. coli expressions of trigramin protein and to de- termine its antitumor activities in vitro. Methods The trigramin gene was synthesized by a slow-annealing PCR method. The recombinant protein was expressed in E. coli system and purified by affinity chromatogra- phy. The control native trigramin was purified from Trimeresurus stejnegeri venom by gel filtration chroma- tography combined with reverse-phase HPLC. The in- hibitory effects of platelet aggregation were measured. Inhibition of cell proliferation or cell motility by tri- gamin were determined by CCK-8 or would healing as-say, respectively. Results The trigramin gene was prepared and expressed in E. coli system. GST-tra- gramin was purified as 89. 1%. The half maximal in- hibitory concentration （IC50） values of platelet aggre- gation by native or recombinant trigramin were 0.35 μmol. L-I or 0.78 μmol · L-l, respectively. Tri- gramin did not affect cell proliferation but inhibited cell motility functions. Conclusion Trigramin gene can be synthesized and then expressed in E. coli system. Trigramin can inhibit cell motility.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2013年第1期36-41,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 30371747)
福建省教育厅科技项目(No 2007F5051)
福建省高等学校建设项目(No XZ04002)
关键词
蛇毒
去整合素
竹叶青素
原核表达
蛋白纯化
细
胞运动
snake venom
disintegrin
trigramin
E.coli expression
protein purification
cell motility
