摘要
为了进一步研究磷脂酶C的生理功能,利用RT-PCR技术扩增了Ds PLC的开放阅读框序列,并与质粒p GS21a连接,构建了原核表达载体p GS21a-Ds PLC。将该重组质粒导入大肠杆菌BL21(DE3)感受态细胞,经IPTG(异丙基-β-D-硫代半乳糖苷)诱导表达出融合蛋白。经SDS-PAGE检测,融合蛋白在包涵体和上清中均存在。可溶性蛋白经His柱纯化后进行电泳分析,结果表明,在96k Da左右有单一的蛋白条带,说明融合蛋白得到有效纯化。蛋白印迹结果显示在96kDa左右有明显的杂交条带,初步证明纯化的蛋白是带有His标签的磷脂酶C。盐藻磷脂酶C的成功表达与纯化,为深入探讨磷脂酶C的性质及功能奠定了基础。
The open reading frame sequence of DsPLC was amplified by RT-PCR technique, and the prokaryotic expression vector pGS21a-DsPLC was constructed with DsPLC and the plasmid pGS21a. Then the recombinant plasmid was introduced into the E. coli BL21 （DE3） competent cells, and the fusion protein was successfully induced by IPTG （lsopropyl - β - D-thiogalactoside）. The fusion protein was presented as the supernatant proteins and inclusion body proteins. The soluble protein was purified by His column and analysed by SDS-PAGE. The result showed that there was a single protein showed that the band about 96KD, obvious hybridizing indicating that the fusion protein was effectively purified. The western blot results band was about 96KD, and it proved that the purified protein was the phospholipase C with His tag. The successful expression and purification of DsPLC has laid sound foundation for further research on the function of DsPLC.
出处
《核农学报》
CAS
CSCD
北大核心
2016年第9期1679-1683,共5页
Journal of Nuclear Agricultural Sciences
基金
国家自然科学基金(31472260
30972240)
关键词
盐藻
磷脂酶C
原核表达
蛋白纯化
蛋白印迹
salina, phospholipase C, prokaryotic expression, protein purification, western blotting
