Construction of a Eukaryotic Expression Vector of Human Cementum Protein 1 and Its Stably Expression in NIH3T3 Cell

摘要

Objective: To construct the recombinant eukaryotic expression vector pcDNA3.1-CEMP1 containing the Human Cementum Protein 1 (hrCEMP1) by PCR and T/A cloning and establish NIH3T3 fibroblast cellline that stably expressing it. Methods: A pair of primers specific for amplifying the DNA fragment encoding hrCEMP1 were designed and synthesized. hrCEMP1 was inserted to the proper sites of vector pcDNA3.1. The recombinant vector pcDNA3.1-hrCEMP1 was propagated in E.coli DH5α, and then was confirmed to contain hrCEMP1 cDNA sequence by agarose gel electrophoresis and DNA sequence analysis. The identified pcDNA3.1-CEMP1 was transfected into NIH3T3 cells by using SofastTM, and stably transfecting cell clones were selected by G418. The transcription of hrCEMP1 in the transfecting cells were determined by RT-PCR. Results: The construction of the recombinant eukaryotic expression vector pcDNA3.1-hrCEMP1 and the correct of the open reading frame were confirmed through restriction enzyme maping analysis and DNA sequencing. RT-PCR results showed there was the special 774bp fragment in the agarose electrophoresis map of the hrCEMP1 gene transfecting cells. Discussion: By PCR and T/A cloning, the cDNA fragment encoding hrCEMP1 can be cloned into pcDNA3.1 to construct the recombinant eukaryotic expression vector pcDNA3.1-hrCEMP1. The NIH3T3 cells that stably expressed hrCEMP1 were screened out.