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人类重组PIF1解螺旋酶C-末端多肽的纯化及其抗体制备 被引量：1

The Purification and Antibody Preparation of C-terminal PIF1 Helicase
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摘要 目的:表达纯化人类PIF1解螺旋酶C-末端的338-641氨基酸的多肽PIF338-641,同时用纯化蛋白制备特异抗体。方法:从HeLa细胞的cDNA文库中PCR扩增得到PIF1解螺旋酶C-末端1012-1926的cDNA片段(对应氨基酸338-641),插入N-末端融合有六聚组氨酸的表达载体pET15b产生pET15b-PIF338-641重组质粒,转化RosettaTM2(DE3)感受态细胞,使PIF338-641蛋白在大肠杆菌中表达,用自制的镍亲和柱纯化PIF338-641蛋白,并以纯化蛋白免疫家兔制备抗血清。结果:PIF338-641蛋白在大肠杆菌中成功表达,纯化了PIF338-641蛋白并制备了PIF338-641蛋白抗血清。结论:制备的PIF338-641蛋白抗体能与蛋白发生特异的免疫反应。 Objective： To purify C - terminal PIF1 heliease, PIF^338-641, with which to make anti - PIF^338-641 - serum. Method： The C - terminal region from 1012 to 1926 base pair eDNA was amplified by PCR from HeLa cell eDNA library and inserted to pET15b with 6x histidine tag at its N - terminus to form pET15b- PIF^338-641 plasmid. The recombinant pET15b- PIF^338-641 plasmid was transformed to RosettaTM 2（DE3 ） and PIF^338 -641 protein was overexpressed. Cohann chromatograph was performed to purify PIF^338- 641 protein by hand- made Ni^2＋ column. The purified PIF^338- 641 was used to immune rabbit to get anti - P1F1 - anti- body. Results： The PIF＆338-641 protein was successfully overexpressed in E. coll. The PIF^338-641 was purified and anti - PIF1 antibody was obtained. Conclusion： anti- PIF1 antibody can produce specific immune reaction with PIF＆338-641 protein.
作者 顾永清 Kenji Kamiya
机构地区 石河子大学医学院 Department of Experimental Oncology
出处 《农垦医学》 2008年第6期449-452,共4页 Journal of Nongken Medicine
基金 supported by a scholarship award from the College Women’s Association of Japan(to GUYongqing)
关键词 PIF1解螺旋酶 蛋白纯化 抗体制备 PIF1 helicase Protein purification antibody
分类号 Q814 [生物学—生物工程]
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参考文献11

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同被引文献9

  • 1MATSON SW,GEORGE JW,BEAN DW.DNA helicases:enzymes with essential roles in all aspects of DNA metabolism[J].Bioessays,1944,16(1):13-22.
  • 2HANADA K,HICKSON ID.Molecular genetics of RecQ helicase disorders[J].Cell Mol Life Sci,2007,64(17):2306-2322.
  • 3BOCHMAN ML,SABOURI N,ZAKIAN VA.Unwinding the functions of the PIF1 family helicases[J].DNA Repair(Amst),2010,9(3):237-249.
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  • 5WANG J,ENGLUND PT,JENSEN RE.TbPIF8,a trypanosoma brucei protein related to the yeast Pif1 helicase,is essential for cell viability and mitochondrial genome maintenance[J].Molmicrobiol,2012,83(3):471-485.
  • 6SCHULDT A.DNA replication:Pif1 overcomes a quadruplex hurdle[J].Nat Rev Mol Cell Biol,2011,12(7):402-408.
  • 7GU Y,MASUDA Y,KAMIYA K.Biochemical analysis of human PIF1 helicase and functions of its N-terminal domain[J].Nucleic Acids Res,2008,39(19):6295-6308.
  • 8MAKOVETS S,BLACKBUM EH.DNA damage signaling prevents deleterious telomere addition at DNA breaks[J].Nat Cell Biol,2009,11(11):1383-1386.
  • 9PIKE JE,BURGERS PM,CAMPBELL JL.Pif1 helicase lengthens some Okazaki fragment flaps necessitating Dna2 nuclease/helicase action in the two nuclease processing pathway[J].J Biol Chem,2009,284(37):25170-25180.

引证文献1

二级引证文献2

农垦医学
2008年 第6期

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