转化生长因子β2真核表达载体pcDNA3.1(+)/TGF-β2的构建及其在骨髓基质细胞的表达

pcDNA3.1(+)/TGF-β2 CONSTRUCTION AND EXPRESSION IN TRANSFECTED MARROW STROMAL CELLS

目的 :构建人转化生长因子 β2的真核表达载体 pcDNA3.1(+) /TGF - β2 ,检测其转染骨髓基质细胞后的表达。方法 :用PCR方法从人胎盘cDNA文库DNA中扩增TGF - β2全长cDNA ,该基因片段两端设计了NheⅠ和XhoⅠ限制性酶切位点。将所得片段连接到T载体上测序证实。利用重组DNA技术将TGF - β2的cDNA构建到真核表达载体 pcDNA3.1(+)上 ,酶切鉴定。通过脂质体介导的转染技术 ,将所构建的载体导入从骨髓中分离培养的基质细胞中 ,体外单层培养。分别于转染后 2 4h和 4周利用RT -PCR和Western -Blotting方法检测目的基因的表达情况。结果 :经琼脂糖凝胶电泳及测序证实 ,PCR获得的片段与GenBank中登记的TGF - β2cDNA序列完全相同。pcDNA3.1(+) /TGF - β2酶切片段的长度与理论大小相符。转染后不同时期的骨髓基质细胞在mRNA和蛋白质水平均可检测到TGF - β2的表达。结论 :pcDNA3.1(+) /TGF - β2真核表达载体构建成功 。

Objective:To construct eukaryotic expression vector of human transforming growth factor β2 (pcDNA3.1/TGF-β2) and verify TGF-β2 expression in pcDNA3.1/TGF-β2 transfected human marrow stromal cells.Methods:Full length coding region of TGF-β2 was amplified by PCR method from human placenta cDNA library DNA.After its sequence was confirmed by sequencing, this cDNA fragment was inserted into a eukaryotic expression vector pcDNA3.1 (+) using gene cloning and DNA recombination. The recombinant was then transferred by liposome to human bone marrow-derived stromal cells. RT-PCR and Western-Blotting were performed to identify the expression of TGF-β2 in transfected cells. Result: The sequence of DNA fragment amplified by PCR is identifical to that published in GenBank. Digestion of the recombinant plasmid with ScaI and NheⅠ plus XhoⅠ liberated DNA fragments with expected size. TGF-β2 was expressed and synthesised by transfected cells both in 24hr and 4w culture interval after transfecion.Conclusion:The eukaryotic expression vector of human transforming growth factor β2 was constructed successfully. Transfection of pcDNA3.1(+)/TGF-β2 is able to provide transient and persistent expression in human marrow stromal cells.