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目的构建pEGFP-N1-BMP2真核表达质粒,并检测其经超声微泡转基因技术转染人牙周膜成纤维细胞(HPDLFs)后的瞬时表达情况。方法从人胎盘滋养层细胞系中提取总RNA,采用RT-PCR方法获得目的基因人骨形成蛋白-2(hBMP2)基因片段,连接pMD19-T载体并测序正确后与真核荧光表达载体pEGFP-N1连接,酶切鉴定后利用超声微泡转基因技术转染入HPDLFs中,通过荧光显微镜和RT-PCR检测目的基因在HPDLFs中的表达。结果成功克隆人BMP2基因,重组质粒pEGFP-N1-BMP2经PCR及双酶切鉴定均证实hBMP2基因已与pEGFP-N1正确重组。pEGFP-N1-BMP2经超声微泡转染HPDLFs后,通过绿色荧光观察和RT-PCR检测证实hBMP2能够在体外培养的HPDLFs内有效的转录和瞬时表达。结论成功构建pEG-FP-N1-BMP2真核表达载体,通过超声微泡转基因技术成功转染入HPDLFs并得到有效表达,为进一步研究该质粒与超声微泡转基因技术在牙周再生基因治疗中的应用提供实验基础。
Objective To construct the eukaryotic expression vector pEGFP-N1-BMP2 and detect the transient expression of pEGFP-N1-BMP2 after it was transfected into human periodontal ligament fibroblasts (HPDLFs) by ultrasound microbubble gene transfection. Methods Total RNA was extracted from human placental trophoblast cell line. The hBMP2 gene fragment was obtained by RT-PCR. The pMD19-T vector was ligated with pMD19-T vector and sequenced. After being identified by restriction enzyme digestion, the cells were transfected into HPDLFs by using ultrasound microbubble gene transfection technology. The expression of the target gene in HPDLFs was detected by fluorescence microscope and RT-PCR. Results The human BMP2 gene was successfully cloned. The recombinant plasmid pEGFP-N1-BMP2 was confirmed by PCR and double enzyme digestion to confirm that hBMP2 gene was correctly recombined with pEGFP-N1. After transfection of pEGFP-N1-BMP2 into HPDLFs by ultrasound microbubbles, hBMP2 could be effectively transcribed and transiently expressed in HPDLFs cultured in vitro by green fluorescence and RT-PCR. CONCLUSION: The eukaryotic expression vector pEG-FP-N1-BMP2 was successfully constructed and successfully transfected into HPDLFs by ultrasound microbubble gene transfection technology. In order to further study the effect of plasmid and ultrasound microbubble gene transfection technology on gene therapy of periodontal regeneration Application provides experimental basis.