利用同源重组构建BMP6、BMPR1B真核表达载体及共转染成纤维细胞表达的研究

Study on Construction of BMP6 and BMPR1B Expression Vectors by Cognate Recombination and Co-transfection of Fibroblasts

旨在构建敖汉细毛羊BMP6、BMPR1B基因的过表达载体,而后将其导入成纤维细胞,分析转染后两基因mRNA、蛋白表达量的变化及基因间相互作用对毛囊的影响。选择40日龄敖汉细毛羊胎羊,采集组织样品,提取RNA后反转录成cDNA,通过PCR反应后利用SoSo试剂盒将纯化的目的片段与表达载体pcDNA3.1连接。酶切鉴定正确后,转至大肠杆菌DH5α感受态细胞,振荡培养并挑取单菌落扩大培养。提取质粒后将pcDNA3.1-BMP6、pcDNA3.1-BMPR1B单、共转染至成纤维细胞中。转染后测定表达量的变化并探究两基因间的作用关系。结果表明,共转染成纤维细胞后BMPR1B基因及BMPR1B蛋白的表达量均较单转染组极显著升高(P<0.01),而BMP6基因及BMP6蛋白的表达量与单转染组无显著差异(P>0.05)。因此,BMP6基因促进了BMPR1B基因的表达,BMPR1B基因对BMP6基因的表达几乎没影响。

The aim of this experiment was to construct overexpression vectors of BMP6 and BMPR1 B genes in Aohan fine-wool sheep,and then introduce them into fibroblast cells,to analyze the changes of mRNA and protein expression of the two genes after transfection and the effect of gene interaction on hair follicle.The 40-day-old embryo of Aohan banner fine-wool sheep was collected,RNA was extracted and reverse transcribed into cDNA.The purified target fragment was linked to PCDNA3.1 by SoSo Kit after PCR reaction.After restriction endonuclease identification,the bacteria was transferred to E.coli DH5αnatural competence,shaken and picked to expand the single colony culture and vibration culture and pick to expand the single colony culture.The plasmids pcDNA3.1-BMP6 and pcDNA3.1-BMPR1 B were co-transfected into fibroblasts.After transfection,the expression level was measured and the relationship between the two genes were explored.The results showed that compared with the single transfected group,the expression of BMPR1 B gene and BMPR1 B protein was significantly increased in co-transfected fibroblasts(P<0.01),but the expression of BMP6 gene and BMP6 protein in co-transfection group had no significant difference(P>0.05).Therefore,BMP6 gene promotes the expression of BMPR1 B gene,and BMPR1 B gene has little effect on the expression of BMP6 gene.