hrCEMP1真核表达载体的构建及其在成纤维细胞株中的表达

Construction of a Eukaryotic Expression Vector of Human Cementum Protein 1 and its Expression in Fibroblast Cell Line

目的:构建含hrCEMP-1基因真核表达质粒。方法:采用PCR方法扩增hrCEMP1基因,并在两端添加合适的酶切位点;利用定向克隆技术将hrCEMP1基因插入到载体pcDNA3.1中,重组的pcDNA3.1-CEMP1在大肠杆菌DH5α内扩增后,通过酶切电泳鉴定和DNA测序证明质粒构建成功;利用高效率的阳离子聚合物,将经过鉴定的pcDNA3.1-CEMP1转染入NIH3T3细胞中,并以RT-PCR检测hrCEMP1基因的转录。结果:通过对重组质粒pcDNA3.1-CEMP1进行酶切鉴定以及DNA序列测定分析,证明真核表达重组质粒pcDNA3.1-CEMP1构建成功,开放阅读框架正确;转染细胞株的RT-PCR结果中观察到特异性242bp片段。结论:成功构建含hrCEMP-1基因真核表达质粒pcDNA3.1-CEMP1,并可在NIH3T3细胞株中mRNA水平表达。

Objective：To construct the recombinant eukaryotic expression vector pcDNA3.1-CEMP1 containing the human cementum protein 1（hrCEMP1） by PCR and T/A cloning. 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-CEMP1 was propagated in E.coli DH5α,and then was confirmed to contain hrCEMP1 cDNA sequence by agarose gel electrophoresis and DNA sequence analysis. The CEMP1 was transfected into NIH3T3 cells by using Sofast TM,and was determined by RT-PCR. Results：The construction of the recombinant eukaryotic expression vector pcDNA3.1-CEMP1 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 242bp fragment in the agarose electrophoresis map of the hrCEMP1 gene transfecting cells. Conclusion：The cDNA fragment encoding hrCEMP1 can be cloned into pcDNA3.1 to construct the recombinant eukaryotic expression vector pcDNA3.1-CEMP1.