摘要
目的采用基因工程技术,在大肠杆菌(E.coli)中表达广西眼镜蛇(黑眼镜蛇)蛇毒神经生长因子(NGF)成熟蛋白并检测其生物活性。方法将天然广西眼镜蛇蛇毒的NGF蛋白基因克隆至融合表达载体pET-40b(+)中,以C端融合了6个组氨酸的形式在E.coli BL21(DE3)通过异丙基硫代半乳糖苷诱导表达NGF蛋白。超声破菌后,将上清液经Ni^(2+)-NTA柱纯化,收集并冻干NGF融合蛋白,用蛋白印迹法分析其NGF抗原活性,并通过促PC12细胞生长法观察其生物活性。结果经蛋白印迹分析可知在38 ku处的条带具有NGF抗原活性。加入天然蛇毒NGF,生长轴突的PC12细胞为(83±3)%,重组品为(21±3)%,表明经纯化的融合蛋白具有NGF生物活力,但活性弱于天然蛇毒NGF。结论在大肠杆菌中可以表达出有活性的蛇毒NGF蛋白。
AIM To express nerve growth factor （NGF） of Naja naja atra （Chinese cobra） in E. coli by genetically engineered method and detect its biological activity. METHODS The eDNA of NGF was cloned into a secretive prokaryotic expression vector pET-40b （ ＋ ）, which carries a C-terminal His Tag sequence. After transforming into E. coli BL21 （ DE3 ）, NGF expression was induced by isopropyl-thio-β-D-galactoside （IPTG）. The expressed product was purified by immobilized metal （ Ni^2 ＋ ） chelation affinity chromatography. The expressed NGF was testified by Western blot. The biological activities of the recombinant NGF and natural NGF were measured by counting the number of PC12 cells with axon. RESULTS The antigen activity of recombinant NGF was expressed by the presenting the 38 ku ladder in the Western blot. The rate of PC12 cells with axon was （83 ±3 ） % in natural NGF, and （21 ±3）% in the recombinant NGF. The results showed that the recombinant NGF has biological activity, but weaker than that of natural NGF. CONCLUSION The NGF with activity can be successfully expressed in E. coll.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2007年第4期265-270,共6页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金(30270304)
广西省科学基金(桂科青0542043)
广西省科学基金(9811039)~~
关键词
眼镜蛇
蛇毒
神经生长因子
蛋白表达
Naja naja atra
cobra venom
nerve growth factor
protein expression
