pcDNA3.1(+)/GDF-5真核表达质粒的构建及其在小鼠骨髓基质干细胞的表达

目的:通过基因重组技术体外构建真核表达质粒pcDNA3．1( )／GDF-5,并检测其在小鼠骨髓基质干细胞中的表达。方法:提取孕14 d小鼠胚胎肢芽组织总RNA,RT-PCR扩增,将扩增产物GDF-5基因片断插入至pcDNA 3．1( )载体,并进行酶切鉴定及测序;脂质体介导pcDNA 3．1( )／GDF-5重组质粒瞬时转染小鼠MSC,RT-PCR和免疫细胞化学检测GDF-5的表达。结果:重组质粒双酶切图谱显示有5．4 kbp和1．6 kbp两条带;测序结果与Genbank中的序列完全相同;转染后RT- PCR显示实验组有一219bp特异性条带,免疫细胞化学检测发现实验组细胞胞浆内有棕色阳性染色,实验对照组和空白组均为阴性。结论:本实验成功构建pcDNA3．1( )／GDF-5真核表达质粒,转染小鼠MSC中有GDF-5表达,为进一步研究其在软骨发育机制和软骨骨组织工程领域提供了实验基础。

Construction of Mammal Expression Plasmid pcDNA 3.1 ( + )/GDF-5 and its Expression in Bone Marrow Mesenchymal Stem Cells of Mice

To construct mammal expression plasmid pcDNA 3.1 ( )/GDF-5 and check the expression of it in bone marrow mesenchyal stem cells of mice. Method: The GDF-5 gene was obtained by RT-PCR from cDNA which was isolated from mouse embryonic limb buds at embryos 14 d, and merged into the pcDNA 3. 1 ( ) vector. The double-stranded DNA of the positive clone was analyzed by restriction endonuclease mapping and DNA sequencing. Then the recombined plasmid was transfected into mouse bone marrow mesenchyal stem cells. Expressions of GDF-5 gene and protein were detected by RT-PCR and immunocytochemistry. Result: Digestion of the recombinant plasmid with double restriction enzyme showed about a 1. 6kbp of specific electrophoretic strip. The se-quence of mouse GDF-5 was consistent with that reported in Genbank. With cells transfected by the expression plasmid pcDNA3. 1 ( ) /GDF-5, expressions of the GDF-5 gene and protein were detected positively, but the untransfected cells were negative. Conclu-sion: The recombinant mammal expression plasmid, pcDNA 3. 1 ( )/GDF-5 can be successfully constructed and expressed in bone marrow mesenchymal stem cells. This provides a basis for further research on GDF-5 gene in the mechanism of chondrogenesis or carti-lage and bone tissue engineering.