利用同源重组技术构建DKK1、BMP4真核表达载体及共转染成纤维细胞表达的研究

Construction of Eukaryotic Expression Vector of DKK1,BMP4 Gene by Homologous Recombination and Its Expression in Fibroblasts

为了构建敖汉细毛羊DKK1、BMP4基因真核表达载体及单、共转染成纤维细胞后研究两基因表达量之间的变化,以敖汉细毛羊为研究对象,采集40日龄的胎羊。首先,通过RNA的提取反转录成c DNA参照Gen Bank中DKK1、BMP4基因序列信息分别设计1对含有同源臂的引物,通过PCR反应扩增获得DKK1、BMP4基因片段、利用So So试剂盒将目的片段连接到pcDNA3. 1真核表达载体上。鉴定正确后的pcDNA3. 1-DKK1、pcDNA3. 1-BMP4重组质粒,转化大肠杆菌DH5α感受态细胞;其次对敖汉细毛羊的成纤维细胞进行分离培养,并将构建的pcDNA3. 1-DKK1、pcDNA3. 1-BMP4单、共转染成纤维细胞,利用荧光定量PCR技术和Western Blot技术检测DKK1、BMP4基因单转染和共转染在成纤维细胞中表达量的变化。结果显示,经酶切、测序鉴定pcDNA3. 1-DKK1、pcDNA3. 1-BMP4构建成功,成纤维细胞原代、传代培养良好,转染后细胞生长良好,经实时荧光定量PCR技术和Western Blot检测DKK1基因共转染成纤维细胞较单转染的表达量极显著下降(P <0. 01),BMP4基因的共转染较单转染表达量没有发生明显变化。成功构建了敖汉细毛羊DKK1、BMP4基因的真核表达载体,并且成功共转染成纤维细胞,DKK1基因的表达量明显下降,BMP4基因的表达量没发生明显变化,因而,推断BMP4基因抑制了DKK1基因的表达,DKK1基因对BMP4基因作用不明显,结果可为进一步研究其功能奠定基础。

The study aimed to use Homologous recombination build DKK1 and BMP4 genes expression vector study the interaction between DKK1 and BMP4 genes by change of expression.Firstly,a pair of primers that have homologous arm were designed by RNA extraction and referred DKK1 and BMP4 genes sequence information of Aohan Fine Wool Sheep in GenBank,the DKK1 and BMP4 genes fragment was amplified by PCR.We used SoSo kit connect the target fragment to pcDNA3.1 eukaryotic expression vector.Recombined plasimid pcDNA3.1-DKK1 and pcDNA3.1-BMP4 was constructed and transformed into E.coli DH5αcompetent cell.Then plasmid pcDNA 3.1-DKK1,BMP4 was cotransfected into fibroblasts,and used qRT-PCR to detect the expression level of these genes.The results showed that,pcDNA3.1-DKK1,BMP4 plasmid cotransfected successfully and identified by enzyme and sequencing.The expression level of DKK1 gene in fibroblasts was lower than the control group and the expression level of BMP4 gene in fibroblasts was higher than the control group.The plasmid was constructed and cotransfected into fibroblasts successfully by Homologous recombination technique.The expression level of DKK1 gene decreased and the expression of BMP4 gene no variation.Therefore,BMP4 gene inhibited the expression of DKK1 gene,and the DKK1 gene had no effect on the expression of BMP4 gene.These results constructed the basis for further research.