【摘要】 背景:有研究表明骨形成蛋白(bone morphogenetic protein,BMP)异源二聚体比同源二聚体的活性高20倍,但相关BMP4/7融合基因的相关病毒载体的构建至今少有报道。目的:构建重组人BMP-4/7融合基因腺相关病毒(adeno-associated virus,AAV)载体并检测其表达。方法:扩增BMP-4、7成熟肽基因;分别构建质粒pGEM-BMP-4和pGEM-BMP-7;采用DNA连接法获取BMP-4/7融合基因,克隆获得pGEM-BMP-4/7质粒,进行基因测序。AAV颗粒转染HEK293细胞,收获病毒载体AAV-BMP-4/7。用SDS-PAGE电泳检测BMP-4/7融合基因蛋白在大肠杆菌的表达。用不同感染复数的AAV-BMP-4/7转染骨髓间充质干细胞3d,提取细胞总RNA和总蛋白,进行RT-PCR和ELISA法检测融合基因BMP-4/7在骨髓间充质干细胞中的表达。结果与结论:PCR电泳,酶切鉴定及测序结果表明,装入pSNAV质粒中的BMP-4/7基因正确,pSNAV-BMP-4/7重组成功。RT-PCR检测到骨髓间充质干细胞BMP-4/7融合基因的转录条带,ELISA检测显示经AAV转染的骨髓间充质干细胞中BMP-4/7蛋白随着感染复数的增加表达逐渐增高(P<0.01)。结果证实,实验成功构建重组骨形成蛋白4/7融合基因腺相关病毒载体。更多还原

【Abstract】 BACKGROUND:Studies have shown that the activity of bone morphogenetic protein(BMP) heterodimer is 20 times higher than that of BMP homodimer,but related BMP 4/7 fusion gene-associated virus vector has been rarely reported.OBJECTIVE:To construct the recombinant adeno-associated virus vector with human BMP 4/7 fusion gene(AAV-BMP-4/7) and to detect its expression.METHODS:Human BMP 4,7 mature peptide gene was amplified.Plasmids pGEM-human BMP 4,7 were constructed,respectively.BMP 4/7 fusion gene was harvested using DNA ligation and then cloned to harvest pGEM-BMP-4/7 plasmid for gene sequencing.HEK293 cells were transfected with adeno-associated virus(AAV) particles to harvest viral vector AAV-BMP-4/7.BMP-4/7 fusion gene protein expression in E.coli was detected by SDS-PAGE.AAV-BMP-4/7 of different multiplicities of infection(MOI) was used to transfect bone marrow stromal cells(BMSCs) for 3 days.Total cellular RNA and total cellular protein were extracted.BMP-4/7 fusion gene expression in BMSCs was detected by RT-PCR and ELISA.RESULTS AND CONCLUSION:The constructed recombinant pSNAV-BMP-4/7 was confirmed by PCR electrophoresis and digestion with restriction enzyme.The gene sequence in the recombinant pSNAV-BMP-4/7 was correct.Ttranscription bands of BMP-4/7 fusion gene in BMSCs were detected by RT-PCR.ELISA showed that BMP-4/7 protein in the AAV-transfected BMSCs was gradually increased with the increase in MOI(P < 0.01).These results showed that the AAV-BMP-4/7 was successfully constructed.更多还原