人同源盒基因HoxB4真核表达载体的构建及鉴定  

Construction and Identification of A Eukaryotic Expression Vector Containing Human Homeobox Gene HoxB4

作　　者：李田兰[1] 赵春亭[1] 张忠广[2] 刘竹珍[1] 刘世海[3] 孟冬梅[4] 张元峰[1] 

机构地区：[1]青岛大学医学院附属医院血液科 [2]青岛大学医学院微生物教研室 [3]青岛大学医学院附属医院中心实验室,山东青岛266003 [4]青岛大学医学院附属医院痛风实验室,山东青岛266003

出　　处：《中国实验血液学杂志》2011年第3期721-724,共4页Journal of Experimental Hematology

基　　金：山东省自然基金项目;编号ZR2009CM056

摘　　要：本研究旨在构建人同源盒基因HoxB4真核表达载体并进行鉴定,为进一步研究HoxB4的作用提供物质基础。采用RT-PCR的方法从健康产妇脐血单个核细胞中扩增出该基因,并将HoxB4的PCR产物用EcoRI和BamHI双酶切后连接到用EcoRI和BamHI双酶切的带有EGFP编码序列和内部核糖体转入位点(IRES)的真核表达载体pIRES2-EGFP中,重组质粒在大肠杆菌DH5α内扩增后,对其进行双酶切及测序鉴定以证实载体构建成功。结果表明,双酶切和质粒测序结果证明HoxB4基因正确的连接到真核表达载体pIRES2-EGFP中,开放阅读框架正确,pIRES2-EGFP/HoxB4真核表达载体构建成功。结论 :利用PCR,DNA重组等分子生物学技术,成功构建了pIRES2-EGFP/HoxB4真核表达载体,为探讨HoxB4基因在造血干细胞的增殖和分化的调控功能提供了实验基础。In order to investigate the special role of HOXB4 in expansion and self renewal of hematopoietic stem cells, the cDNA of HOXB4 was extracted and cloned from umbilical cord blood mononucler cells by using RT-PCR . Then the eukaryotic expression bicistron plasmid vector plRES2-EGFP/HOXB4 was designed and constructed after cutting HOXB4 and plRES2-EGFP respectively by restriction enzyme EcoRI and BamHI. The recombinant plasmid was delivered into competent cells of Escherichia coli. The successful construction of plasmid was confirmed by the identification of endonuclease cutting and sequencing. The results showed that the HOXB4 cDNA was cloned successfully from umbilical cord blood mononucler cells and the recombinant eukaryotic expression bicistron plasmid vector was constructed, and then introduced it into 293T cells successfully. It is concluded that a plRES2-EGFP/HoxB1 eukaryotic expression bicistron plasmid vector has been constructed successfully, which results provide a useful material basis for exploration of HoxB4 function in the proliferation and differentiation of hematopoietic cells.

关 键 词：同源盒基因 HOXB4 重组质粒 真核表达载体 

分 类 号：Q789[生物学—分子生物学] Q782