摘要
目的制备重组人SMAC及SMAC-PTD。方法经RT-PCR从人HeLa细胞中扩增人野生型SMAC及SMAC-PTD cDNA,分别构建于原核表达质粒pET28 a(+),测序正确后,将重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,N i2+-NTA层析纯化,SDS-PAGE和W estern b lot鉴定目的蛋白。结果RT-PCR扩增分别得到编码人SMAC及SMAC-PTD cDNA片断,测序结果正确,重组蛋白在大肠杆菌中获得了高效表达。结论成功制备了高纯度的人野生型SMAC及SMAC-PTD融合蛋白,为进一步研究奠定了基础。
Objective To prepare human second mitochondrial derived activator of caspase （hSMAC） and human SMAC-PTD （hSMAC-PTD）. Methods The eDNA fragments encoding hSMAC and hSMAC-PTD were amplified from HeLa ceils by reverse transcription polymerase chain reaction （RT-PCR） and PCR. PCR products were cloned into the prokaryotic expression vector pET28a （ ＋ ） for sequencing. The vectors with correct sequence were transformed into E coli. BL21 （DE3） to express the hSMAC and hSMAC-PTD fusion protein with the induction of IPTG. The hSMAC and hSMAC-PTD fusion protein were purified with Ni^2＋ -NTA chromatography, and identified by SDS-PAGE and Western blotting. Results The eDNA fragments encoding hSMAC and hSMAC-PTD were amplified by RT-PCR and PCR were accurate. The hSMAC-PTD and hSMAC were highly expressed and purified. Conclusion The eDNA fragment encoding human SMAC and SMAC-PTD has been successfully cloned, and the proteins are highly expressed and purified.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第9期958-960,共3页
Journal of Third Military Medical University
