Construction of a Eukaryotic Expression Vector of Human Cementum Protein 1 and its Stably expression in NIH3T3 cell
Objective: To construct the recombinant eukaryoticexpression vector pcDNA3.1-CEMP1 containing the HumanCementum Protein 1 (hrCEMP1) by PCR and T/A cloning andestablish NIH3T3 fibroblast cellline that stably expressing it.Methods: A pair of primers specific for amplifying the DNAfragment encoding hrCEMP1 were designed and synthesized.hrCEMP1 was inserted to the proper sites of vector pcDNA3.1. Therecombinant vector pcDNA3.1-hrCEMP1 was propagated in E.coliDH5 α , and then was confirmed to contain hrCEMP1 cDNA sequenceby agarose gel electrophoresis and DNA sequence analysis. Theidentified pcDNA3.1-CEMP1 was transfected into NIH3T3 cells byusing Sofast TM , and stably transfecting cell clones were selected byG418. The transcription of hrCEMP1 in the transfecting cells weredetermined by RT-PCR. Results: The construction of therecombinant eukaryotic expression vector pcDNA3.1-hrCEMP1 andthe correct of the open reading frame were confirmed throughrestriction enzyme maping analysis and DNA sequencing. RT-PCRresults showed there was the special 774bp fragment in the agaroseelectrophoresis map of the hrCEMP1 gene transfecting cells.Discussion: By PCR and T/A cloning, the cDNA fragment encodinghrCEMP1 can be cloned into pcDNA3.1 to construct the recombinanteukaryotic expression vector pcDNA3.1-hrCEMP1. The NIH3T3cells that stably expressed hrCEMP1 were screened out.
Yu Liu Weibin Sun
School of Stomatology,Nanjing University, Medical Center Nanjing, China
国际会议
成都
英文
1-4
2010-06-18（万方平台首次上网日期，不代表论文的发表时间）