可表达骨形态发生蛋白-4的真核表达载体的构建与鉴定

Construction of a eukaryotic expression vector including the target gene fragment hBMP-4

目的构建含有骨形态发生蛋白(hBMP)-4的真核表达载体pcDNA3·1(+)/hBMP-4。方法将质粒pCS2(+)/hBMP-4用EcoRⅠ单酶双切获得目的基因片段,真核载体pcDNA3·1(+)用EcoRⅠ酶切线性化,对上述两种酶切产物分别回收纯化后进行连接,筛选出正确的转化子,并进一步转染COS-7细胞,提取总RNA和总蛋白,进行RT-PCR和Western-blot检测。结果获得了含有目的基因片段的真核表达载体,酶切及基因测序提示构建正确,RT-PCR检测到目的基因的转录,Western-blot检测到hBMP-4蛋白表达。结论利用非定向克隆技术将目的基因转移至真核表达载体中,为进一步研究目的基因打下基础。

Objective To construct a eukaryotic expression vector including the target gene fragment hBMP-4（human bone morphogenetic protein）. Methods hBMP-4 gene fragment was cut down by EcoR Ⅰ from pCS2（ ＋ ）/ hBMP-4 and then subeloned into eukaryotic expressive vector pcDNA 3. 1 （ ＋ ）. The resulting recombinant plasmid pcDNA 3.1 （ ＋ ）/hBMP-4 was transiently tranfected into COS-7 cells using Lipofectamine-2000. The resulting plasmid was analyzed using RT-PCR and Western-blot 72 hours later. Results The recombinant plasmid of pcDNA 3. 1 （ ＋ ）/hBMP-4 was confirmed by restriction endonuclease digestion and sequencing; the specific transcription and expression of hBMP-4 protein in COS-7 cells was confirmed by RT-PCR and Western-blot assay. Conclusion The eukaryotic expression vector containing hBMP-4 gene has been constructed and the fusion protein is expressed successfully. This has laid a foundation for further research of gene-therapy of hBMP-4.