人PIF1及其截短体重组蛋白的真核表达及其细胞内定位  

The eukaryotic expression and intracellular location of human PIF1 and its truncated recombinant protein

作　　者：李珊珊[1] 刘旭[1] 张莹[1] 王建校[1] 刘晓丹 王豫[2] 周平坤[2] 顾永清[1,2] 

机构地区：[1]石河子大学医学院,新疆石河子832003 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《西安交通大学学报（医学版）》2014年第5期595-598,699,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：国家自然科学基金资助项目(No.30960093;31160183;31270894)~~

摘　　要：目的构建人PIF1及其C末端解螺旋酶模序(PIF1C)和N末端PINT结构域(PIF1N)截短体真核表达重组质粒,进行真核表达,并确定PIF1、PIF1C、PIF1N在细胞内定位。方法以HeLa cDNA为模板PCR获取PIF1、PIF1C、PIF1N编码区cDNA,将其插入pCMV-Tag 2B质粒中构建PIF1、PIF1C、PIF1N真核表达重组质粒;通过lipofectamine2000将重组质粒转染人293细胞中,Western blot方法检测其在真核细胞内的表达,用免疫荧光法检测其细胞内定位。结果成功构建了PIF1及其截短体真核表达重组体,并在真核细胞内成功表达。确定PIF1定位于核内,PIF1C定位于整个细胞,呈弥散分布,PIF1N主要定位于核内。结论在真核细胞中成功表达了PIF1、PIF1C、PIF1N,且发现PIF1N对PIF1的入核起重要作用,为进一步研究其PIF1及其各结构域奠定了实验基础。Objective To construct the recombinant eukaryotic expression plasmids of human PIF1,its C terminal helicase motif （PIF1C） and N terminal PINT domain （PIF1N）,and to explore the intracellular location of PIF1,PIF1C and PIF1N.Methods By using Hela cDNA as template,the cDNAs encoding PIF1,PIF1C and PIF1N were amplified by PCR and inserted into pCMV-Tag 2B plasmid to construct recombinant plasmids named PIF1,PIF1C and PIF1N.The recombinant plasmids were transfected into 293 cells using lipofectamine 2000.The protein expressions and the intracellular locations of the inserted genes were confirmed by Western blot and immunofluorescence,respectively.Results The recombinant eukaryotic expression plasmid of human PIF1 and its truncated mutants were successfully constructed and transfected into eukaryote cells.PIF1 was located in the nucleus and PIF1C was found in the whole cell and was dispersively distributed,while PIF1N was located in the nucleus.Conclusion PIF1,PIF1C and PIF1N were successfully expressed in eukaryote cells.PIF1N plays a central role in the nucleus entrance of PIF1.The present study provides important experimental evidence for further study on PIF1 and its domains.

关 键 词：解螺旋酶 PIF1 重组质粒 蛋白表达 免疫荧光 真核表达 

分 类 号：Q78[生物学—分子生物学]