6种转录本人早幼粒细胞性白血病基因真核表达质粒的构建及其重组蛋白的亚细胞定位分析

Construction of eukaryotic expression plasmid of human promyelocytic leukemia gene of six transcripts and subcellular location analysis of its recombinant proteins

目的 构建6种转录本人早幼粒细胞性白血病(human promyelocytic leukaemia,hPML)基因的真核表达质粒,并分析其重组蛋白的亚细胞定位情况。方法 根据GenBank中登录的hPML基因序列设计引物,RT-PCR法扩增获得6种转录本hPML基因片段(hPMLⅠ、Ⅱ、Ⅳ、Ⅴ、Ⅵ、Ⅶ),分别连接至真核表达载体pCAGGS,获得6种转录本hPML基因的真核表达质粒。将6种真核表达质粒分别转染293T细胞,Western blot法检测蛋白表达情况;分别转染至Vero细胞,间接免疫荧光(indirect immunofluorescence assay,IFA)法检测亚细胞定位情况。结果 6种真核表达质粒中的目的基因片段均与GenBank中登录的hPML基因序列一致。6种重组蛋白均可与Myc抗体发生特异性结合,其中重组蛋白hPMLⅠ、Ⅱ、Ⅳ、Ⅴ和Ⅵ定位于细胞核及细胞质内,重组蛋白hPMLⅦ主要定位于细胞质,极少定位于细胞核中。结论构建的6种转录本hPML基因真核表达质粒均可在哺乳动物细胞中正确表达,表达的重组蛋白同时定位于细胞核及细胞质或主要定位于细胞质中。本研究为重组蛋白hPML抗病毒等生物学功能的深入研究提供了实验依据。

Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.