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目的构建真核表达载体p Flag-dlx3,并将其转染成牙本质样细胞株i MDP3,检测外源dlx3基因在i MDP3细胞内的表达。方法提取新生小鼠牙髓总RNA,RT-PCR扩增Dlx3目的基因片段,并将该片段克隆至PCR-TopoⅡ载体中;经鉴定正确后目的基因与表达载体p Flag-CMV连接;Lipofectamin 2000介导重组质粒p Flag-dlx3转染i MDP3;Western-blot鉴定外源性dlx3在细胞内的表达。结果重组p Flag-dlx3质粒经酶切、测序鉴定正确;i MDP3细胞内检测到外源dlx3蛋白的表达;转染p Flag-dlx3组dlx3蛋白的表达量显著高于正常组。结论成功构建真核表达载体p Flag-dlx3,且外源dlx3基因可在i MDP3细胞内过表达。
Objective To construct eukaryotic expression vector p Flag-dlx3 and transfect it into dentin-like cell line i MDP3 to detect the expression of exogenous dlx3 gene in i MDP3 cells. Methods Total RNA was extracted from dental pulp of newborn mice. The target gene fragment of Dlx3 was amplified by RT-PCR and cloned into PCR-TopoⅡ vector. After identification, the target gene was ligated to p Flag-CMV. Lipofectamine 2000 Recombinant plasmid p Flag-dlx3 transfected i MDP3; Western-blot identification of exogenous dlx3 expression in the cell. Results The recombinant plasmid p Flag-dlx3 was identified by restriction enzyme digestion and sequencing. The expression of exogenous dlx3 protein was detected in i MDP3 cells. The expression of dlx3 protein in transfected p Flag-dlx3 group was significantly higher than that in normal group. Conclusion The eukaryotic expression vector p Flag-dlx3 was successfully constructed and the exogenous dlx3 gene was overexpressed in i MDP3 cells.