摘要
解螺旋酶参与几乎体内所有的DNA代谢，具有重要的生理功能。为了从分子水平阐述人类PIF1解螺旋酶的生理功能，我们以HeLa细胞的cDNA文库为模版，PCR扩增得到PIF1基因5’端含有1～534核苷酸的cDNA序列一PIFAC。在PIF△C的5’端引入六组氨酸标签后插入pET15b表达载体，得到重组质粒pET15-PIF△C。以此重组质粒转化Rosetta TM2（DE3）感受态细胞，使PIFAC蛋白质在大肠杆菌中得到表达。在4℃通过快速液相色谱纯化系统，通过一系列色谱层析柱纯化了PIFAC蛋白质。以纯化的PIFAC蛋白质免疫家兔制备了抗血清，并检测了纯化的PIFAC的生物化学活性。结果显示不含解旋酶模序的人类PIF1蛋白质的N-末端，具有使单链DNA复性的特性。PIF1解螺旋酶具有解开DNA双链和使双链DNA复性这一矛盾的特性，暗示了人类PIF1解螺旋酶可能参与损伤DNA的修复，包括断裂的双链DNA的修复。
Helieases participate almost in all the DNA metabolism, therefore have important physiological function. In order to elucidate cellular function of human PIF1 helicase from the molecular level, the 5＇region of PIF1 cDNA, from 1 - 534 nucleotides,was amplified by PCR using the HeLa cell eDNA library as template. The eDNA with a histidine tag at the N-terminus was inserted into the pET15b vector to produce recombinant plasmid, pET15-PIFAC. The recombinant PIFAC was overexpressed in RosettaTM 2（ DE3 ）. At 4℃ through a series of affinity column ,the re- combinant PIFAC protein was purified by fast protein liquid chromatograph （FPLC）. The purified PIFAC was used to immune rabbit to get anti-PIFl-serum and the biochemical activity of PIFAC was determined. Our results showed N-terminal region of human PIF1 helicase,which excluded helicase motifs,had single strand DNA annealing activi- ty. That PIFI helicase has apparently antagonistic unwinding and annealing activity indicates it may participate in repairing of damaged DNA,including repairing double-strand broken DNA.
出处
《石河子大学学报（自然科学版）》
CAS
2009年第2期139-143,共5页
Journal of Shihezi University(Natural Science)
